US20040254130A1 - Chlamydia antigens and corresponding dna fragments and uses thereof - Google Patents

Chlamydia antigens and corresponding dna fragments and uses thereof Download PDF

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US20040254130A1
US20040254130A1 US10/275,652 US27565203A US2004254130A1 US 20040254130 A1 US20040254130 A1 US 20040254130A1 US 27565203 A US27565203 A US 27565203A US 2004254130 A1 US2004254130 A1 US 2004254130A1
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polypeptide
nucleic acid
acid sequence
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Andrew Murdin
Raymond Oomen
Joe Wang
Pamela Dunn
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Sanofi Pasteur Ltd
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Aventis Pasteur Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/295—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Chlamydiales (O)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a number of Chlamydia antigens, including an ATP-binding cassette protein, a secretory locus ORF, an endopeptidase, a protease, a metalloprotease, CLP protease ATPase, a CLP protease subunit, a translycolase/transpeptidase, a CLPc protease and thioredoxin, and their corresponding DNA molecules, for the prevention and treatment of Chlamydia infection in mammals.
  • an ATP-binding cassette protein including an ATP-binding cassette protein, a secretory locus ORF, an endopeptidase, a protease, a metalloprotease, CLP protease ATPase, a CLP protease subunit, a translycolase/transpeptidase, a CLPc protease and thioredoxin, and their corresponding DNA molecules, for the
  • Chlamydiae are prokaryotes. They exhibit morphologic and structural similarities to gram-negative bacteria including a trilaminar outer membrane, which contains lipopolysaccharide and several membrane proteins that are structurally and functionally analogous to proteins found in E coli . They are obligate intra-cellular parasites with a unique biphasic life cycle consisting of a metabolically inactive but infectious extracellular stage and a replicating but non-infectious intracellular stage. The replicative stage of the life-cycle takes place within a membrane-bound inclusion which sequesters the bacteria away from the cytoplasm of the infected host cell.
  • C. pneumoniae is a common human pathogen, originally described as the TWAR strain of Chlamydia psittaci but subsequently recognised to be a new species.
  • C. pneumoniae is antigenically, genetically and morphologically distinct from other Chlamydia species ( C. trachomatis, C. pecorum and C. psittaci ). It shows 10% or less DNA sequence homology with either of C. trachomatis or C. psittaci.
  • C. pneumoniae is the third most common cause of community acquired pneumonia, only less frequent than Streptococcus pneumoniae and Mycoplasma pneumoniae (Grayston et al. (1995) Journal of Infectious Diseases 168:1231; Campos et al. (1995) Investigation of Ophthalmology and Visual Science 36:1477). It can also cause upper respiratory tract symptoms and disease, including bronchitis and sinusitis (Grayston et al. (1995) Journal of Infectious Diseases 168:1231; Grayston et al (1990) Journal of Infectious Diseases 161:618-625; Marrie (1993) Clinical Infectious Diseases.
  • C. pneumoniae infection usually presents as an acute respiratory disease (i.e., cough, sore throat, hoarseness, and fever; abnormal chest sounds on auscultation). For most patients, the cough persists for 2 to 6 weeks, and recovery is slow. In approximately 10% of these cases, upper respiratory tract infection is followed by bronchitis or pneumonia. Furthermore, during a C. pneumoniae epidemic, subsequent co-infection with pneumococcus has been noted in about half of these pneumonia patients, particularly in the infirm and the elderly. As noted above, there is increasing evidence that C. pneumoniae infection is also linked to diseases other than respiratory infections.
  • C. pneumoniae appears to spread slowly through a population (case-to-case interval averaging 30 days) because infected persons are inefficient transmitters of the organism. Susceptibility to C. pneumoniae is universal. Reinfections occur during adulthood, following the primary infection as a child.
  • pneumoniae appears to be an endemic disease throughout the world, noteworthy for superimposed intervals of increased incidence (epidemics) that persist for 2 to 3 years.
  • C. trachomatis infection does not confer cross-immunity to C. pneumoniae .
  • Infections are easily treated with oral antibiotics, tetracycline or erythromycin (2 g/d, for at least 10 to 14 d).
  • a recently developed drug, azithromycin is highly effective as a single-dose therapy against chlamydial infections.
  • C. pneumoniae infection is often mild and without complications, and up to 90% of infections are subacute or unrecognized.
  • infections have been thought to be rare up to the age of 5 y, although a recent study (E Normann et al, Chlamydia pneumoniae in children with acute respiratory tract infections, Acta Paediatrica, 1998, Vol 87, Iss 1, pp 23-27) has reported that many children in this age group show PCR evidence of infection despite being seronegative, and estimates a prevalence of 17-19% in 2-4 y olds.
  • the seroprevalence of C. pneumoniae antibodies among young children is elevated, and there are suspicions that C. pneumoniae may be an important cause of acute lower respiratory tract disease and mortality for infants and children in tropical regions of the world.
  • C. pneumoniae infection usually happens between the ages of 5 and 20 y. In the USA, for example, there are estimated to be 30,000 cases of childhood pneumonia each year caused by C. pneumoniae . Infections may cluster among groups of children or young adults (e.g., school pupils or military conscripts).
  • C. pneumoniae infection may progress to chronic bronchitis and sinusitis.
  • a study in the USA revealed that the incidence of pneumonia caused by C. pneumoniae in persons younger than 60 years is 1 case per 1,000 persons per year; but in the elderly, the disease incidence rose three-fold.
  • C. pneumoniae infection rarely leads to hospitalization, except in patients with an underlying illness.
  • a disease associated with C. trachomatis infection is trachoma, a sequela of ocular infection. This disease continues to be a major cause of preventable blindness, with an estimated 500 million cases of active trachoma worldwide (seven million include blindness from conjunctival scarring and eyelid deformities).
  • genital chlamydial infection has been identified as a major public health problem because of the recognition that chlamydial infection is associated with disease syndromes such as non-gonococcal urethritis, mucopurulent cervicitis, pelvic inflammatory disease (PID), ectopic pregnancy, and tubal infertility.
  • PID pelvic inflammatory disease
  • the World Health Organization estimated 89 million new cases of genital chlamydial infections worldwide in 1995. In the United States, each year an estimated four million new cases occur and 50,000 women become infertile as a result of infection.
  • mice which have recovered from a lung infection with C. trachomatis are protected from infertility induced by a subsequent vaginal challenge (Pal et al. (1996) Infection and Immunity.64:5341).
  • sheep immunized with inactivated C. psittaci were protected from subsequent chlamydial-induced abortions and stillbirths (Jones et al. (1995) Vaccine 13:715).
  • the gene encoding a 76 kDa antigen has been cloned from a single strain of C. pneumoniae and the sequence published (Perez Melgosa et al. Infection and Immunity.(1994) 62:880).
  • An operon encoding the 9 kDa and 60 kDa cyteine-rich outer membrane protein genes has been described (Watson et al., Nucleic Acids Res (1990) 18:5299; Watson et al., Microbiology (1995) 141:2489).
  • Many antigens recognized by immune sera to C. pneumoniae are conserved across all chlamydiae , but 98 kDa, 76 kDa and several other proteins may be C.
  • the present invention provides purified and isolated polynucleotide molecules that encode a Chlamydia polypeptide selected from: an ATP-binding cassette protein, a secretory locus ORF, an endopeptidase, a protease, a metalloprotease, CLP protease ATPase, a CLP protease subunit, a translycolase/transpeptidase, a CLPc protease and thioredoxin.
  • the polynucleotide molecules can be used in methods to prevent, treat, and diagnose Chlamydia infection.
  • the polynucleotide molecules is DNA that encode a polypeptide of any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
  • Another form of the invention provides polypeptides corresponding to an isolated DNA molecule. Amino acid sequences of the corresponding encoded polypeptides are shown in one embodiment as SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
  • the invention having provided the polynucleotide sequences encoding Chlamydia polypeptides, also provides polynucleotides encoding fragments derived from such polypeptides. Moreover, the invention is understood to provide mutants and derivatives of such polypeptides and fragments derived therefrom, which result from the addition, deletion, or substitution of non-essential amino acids as described herein. Those skilled in the art would also readily understand that the invention, having provided the polynucleotide sequences encoding Chlamydia polypeptides, further provides monospecific antibodies that specifically bind to such polypeptides.
  • the present invention has wide application and includes expression cassettes, vectors, and cells transformed or transfected with the polynucleotides of the invention. Accordingly, the present invention further provides (i) a method for producing a polypeptide of the invention in a recombinant host system and related expression cassettes, vectors, and transformed or transfected cells; (ii) a vaccine, or a live vaccine vector such as a pox virus, Salmonella typhimurium , or Vibrio cholerae vector, containing a polypeptide or a polynucleotide of the invention, such vaccines and vaccine vectors being useful for, e.g., preventing and treating Chlamydia infection, in combination with a diluent or carrier, and related pharmaceutical compositions and associated therapeutic and/or prophylactic methods; (iii) a therapeutic and/or prophylactic use of an RNA or DNA molecule of the invention, either in a naked form or formulated with a delivery vehicle, a poly
  • One aspect of the invention provides a vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
  • nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19;
  • each first nucleic acid is capable of being expressed.
  • Another aspect of the invention provides a vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
  • nucleic acid sequence comprising at least 36 consecutive nucleotides from any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19;
  • nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20;
  • each first nucleic acid is capable of being expressed.
  • FIG. 1 shows the nucleotide sequence of the gene encoding an ATP-binding cassette (SEQ ID No: 1) and the deduced amino acid sequence of the ATP-binding cassette from Chlamydia pneumoniae (SEQ ID No: 2).
  • FIG. 2 shows the nucleotide sequence of the gene encoding a secretory locus ORF (SEQ ID No: 3) and the deduced amino acid sequence of the secretory locus ORF from Chlamydia pneumoniae (SEQ ID No: 4).
  • FIG. 3 shows the nucleotide sequence of the gene encoding an endopeptidase (SEQ ID No: 5) and the deduced amino acid sequence of the endopeptidase from Chlamydia pneumoniae (SEQ ID No: 6).
  • FIG. 4 shows the nucleotide sequence of the gene encoding a protease (SEQ ID No: 7) and the deduced amino acid sequence of the protease from Chlamydia pneumoniae (SEQ ID No: 8).
  • FIG. 5 shows the nucleotide sequence of the gene encoding a metalloprotease (SEQ ID No: 9) and the deduced amino acid sequence of the metalloprotease from Chlamydia pneumoniae (SEQ ID No: 10).
  • FIG. 6 shows the nucleotide sequence of the gene encoding CLP protease ATPase (SEQ ID No: 11) and the deduced amino acid sequence of the CLP protease ATPase from Chlamydia pneumoniae (SEQ ID No: 12).
  • FIG. 7 shows the nucleotide sequence of the gene encoding a CLP protease subunit (SEQ ID No: 13) and the deduced amino acid sequence of the CLP protease subunit from Chlamydia pneumoniae (SEQ ID No: 14).
  • FIG. 8 shows the nucleotide sequence of the gene encoding a translycolase/transpeptidase (SEQ ID No: 15) and the deduced amino acid sequence of the transglycolase/transpeptidase from Chlamydia pneumoniae (SEQ ID No: 16).
  • FIG. 9 shows the nucleotide sequence of the gene encoding a CLPc protease (SEQ ID No: 17) and the deduced amino acid sequence of the CLPc protease from Chlamydia pneumoniae (SEQ ID No: 18).
  • FIG. 10 shows the nucleotide sequence of the gene encoding thioredoxin (SEQ ID No: 19) and the deduced amino acid sequence of thioredoxin from Chlamydia pneumoniae (SEQ ID No: 20).
  • FIG. 11 shows the restriction enzyme analysis of the C. pneumoniae gene encoding an ATP-binding cassette.
  • FIG. 12 shows shows the restriction enzyme analysis of the C. pneumoniae gene encoding a secretory locus ORF.
  • FIG. 13 shows the restriction enzyme analysis of the C. pneumoniae gene encoding an endopeptidase.
  • FIG. 14 shows the restriction enzyme analysis of the C. pneumoniae gene encoding a protease.
  • FIG. 15 shows the restriction enzyme analysis of the C. pneumoniae gene encoding a metalloprotease.
  • FIG. 16 shows the restriction enzyme analysis of the C. pneumoniae gene encoding CLP protease ATPase.
  • FIG. 17 shows the restriction enzyme analysis of the C. pneumoniae gene encoding a CLP protease subunit.
  • FIG. 18 shows the restriction enzyme analysis of the C. pneumoniae gene encoding a translycolase/transpeptidase.
  • FIG. 19 shows the restriction enzyme analysis of the C. pneumoniae gene encoding a CLPc protease.
  • FIG. 20 shows the restriction enzyme analysis of the C. pneumoniae gene encoding thioredoxin.
  • FIG. 21 shows the construction and elements of plasmid pCACPNM213.
  • FIG. 22 shows the construction and elements of plasmid pCACPNM882.
  • FIG. 23 shows the construction and elements of plasmid pCACPNM208.
  • FIG. 24 shows the construction and elements of plasmid pCACPNM1096.
  • FIG. 25 shows the construction and elements of plasmid pCACPNM1097.
  • FIG. 26 shows the construction and elements of plasmid pCACPNM908.
  • FIG. 27 shows the construction and elements of plasmid pCACPNM909.
  • FIG. 28 shows the construction and elements of plasmid pCACPNM440.
  • FIG. 29 shows the construction and elements of plasmid pCACPNM459.
  • FIG. 30 shows the construction and elements of plasmid pCACPNM708.
  • FIG. 31 illustrates protection against C. pneumoniae infection by pCACPNM213 following DNA immunization.
  • FIG. 32 illustrates protection against C. pneumoniae infection by pCACPNM882 following DNA immunization.
  • FIG. 33 illustrates protection against C. pneumoniae infection by pCACPNM208 following DNA immunization.
  • FIG. 34 illustrates protection against C. pneumoniae infection by pCACPNM1096 following DNA immunization.
  • FIG. 35 illustrates protection against C. pneumoniae infection by pCACPNM1097 following DNA immunization.
  • FIG. 36 illustrates protection against C. pneumoniae infection by pCACPNM908 following DNA immunization.
  • FIG. 37 illustrates protection against C. pneumoniae infection by pCACPNM909 following DNA immunization.
  • FIG. 38 illustrates protection against C. pneumoniae infection by pCACPNM2440 following DNA immunization.
  • FIG. 39 illustrates protection against C. pneumoniae infection by pCACPNM459 following DNA immunization.
  • FIG. 40 illustrates protection against C. pneumoniae infection by pCACPNM708 following DNA immunization.
  • ORFs Open reading frames encoding a number of Chlamydial proteins have been identified from the C. pneumoniae genome. These proteins include an ATP-binding cassette protein, a secretory locus ORF, an endopeptidase, a protease, a metalloprotease, CLP protease ATPase, a CLP protease subunit, a translycolase/transpeptidase, a CLPc protease and thioredoxin. The gene encoding each of these polypeptides has been inserted into an expression plasmid and shown to confer immune protection against chlamydial infection. Accordingly, any one of these and related polypeptides can be used to prevent and treat Chlamydia infection.
  • isolated polynucleotides which encode Chlamydia polypeptides, whose amino acid sequences are shown in SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
  • isolated polynucleotide is defined as a polynucleotide removed from the environment in which it naturally occurs.
  • a naturally-occurring DNA molecule present in the genome of a living bacteria or as part of a gene bank is not isolated, but the same molecule separated from the remaining part of the bacterial genome, as a result of, e.g., a cloning event (amplification), is isolated.
  • an isolated DNA molecule is free from DNA regions (e.g., coding regions) with which it is immediately contiguous at the 5′ or 3′ end, in the naturally occurring genome.
  • Such isolated polynucleotides may be part of a vector or a composition and still be defined as isolated in that such a vector or composition is not part of the natural environment of such polynucleotide.
  • the polynucleotide of the invention is either RNA or DNA (cDNA, genomic DNA, or synthetic DNA), or modifications, variants, homologs or fragments thereof.
  • the DNA is either double-stranded or single-stranded, and, if single-stranded, is either the coding strand or the non-coding (anti-sense) strand.
  • any one of the sequences that encode the polypeptides of the invention as shown in any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 is (a) a coding sequence, (b) a ribonucleotide sequence derived from transcription of (a), or (c) a coding sequence which uses the redundancy or degeneracy of the genetic code to encode the same polypeptides.
  • polypeptide or “protein” is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Both terms are used interchangeably in the present application.
  • amino acid sequences are provided which are homologous to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
  • “homologous amino acid sequence” is any polypeptide which is encoded, in whole or in part, by a nucleic acid sequence which hybridizes at 25-35° C. below critical melting temperature (Tm), to any portion of the nucleic acid sequence of any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19.
  • Tm critical melting temperature
  • a homologous amino acid sequence is one that differs from an amino acid sequence shown in any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 by one or more conservative amino acid substitutions.
  • Such a sequence also encompass serotypic variants (defined below) as well as sequences containing deletions or insertions which retain inherent characteristics of the polypeptide such as immunogenicity.
  • a sequence is at least 75%, preferably at least 78%, more preferably at least 80%, even more preferably at least 85%, 88% or 90%, and most preferably at least 93%, 95% or 98% identical to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
  • Homologous amino acid sequences include sequences that are identical or substantially identical to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
  • amino acid sequence substantially identical is meant a sequence that is at least 90%, preferably 95%, more preferably 97%, and most preferably 99% identical to an amino acid sequence of reference and that preferably differs from the sequence of reference by a majority of conservative amino acid substitutions.
  • Conservative amino acid substitutions are substitutions among amino acids of the same class. These classes include, for example, amino acids having uncharged polar side chains, such as asparagine, glutamine, serine, threonine, and tyrosine; amino acids having basic side chains, such as lysine, arginine, and histidine; amino acids having acidic side chains, such as aspartic acid and glutamic acid; and amino acids having nonpolar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine.
  • amino acids having uncharged polar side chains such as asparagine, glutamine, serine, threonine, and tyrosine
  • amino acids having basic side chains such as lysine, arginine, and histidine
  • amino acids having acidic side chains such as aspartic acid and glutamic acid
  • amino acids having nonpolar side chains such
  • sequence analysis software such as Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705. Amino acid sequences are aligned to maximize identity. Gaps may be artificially introduced into the sequence to attain proper alignment. Once the optimal alignment has been set up, the degree of homology is established by recording all of the positions in which the amino acids of both sequences are identical, relative to the total number of positions.
  • homologous polynucleotide sequences are defined in a similar way.
  • a homologous sequence is one that is at least 45%, more preferably 50%, 55%, 60%, 65%, 70%, 75%, 80%, and even more preferably 85%, 87%, 90%, 93%, 96% and most preferably 99% identical to the coding sequence of any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19.
  • polypeptides having a sequence homologous to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 include naturally-occurring allelic variants, as well as mutants or any other non-naturally occurring variants that retain the inherent characteristics of the polypeptide of any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
  • an allelic variant is an alternate form of a polypeptide that is characterized as having a substitution, deletion, or addition of one or more amino acids that does not alter the biological function of the polypeptide.
  • biological function is meant the function of the polypeptide in the cells in which it naturally occurs, even if the function is not necessary for the growth or survival of the cells.
  • biological function of a porin is to allow the entry into cells of compounds present in the extracellular medium.
  • Biological function is distinct from antigenic property.
  • a polypeptide can have more than one biological function.
  • Allelic variants are very common in nature.
  • a bacterial species such as C. pneumoniae
  • C. pneumoniae is usually represented by a variety of strains that differ from each other by minor allelic variations.
  • a polypeptide that fulfills the same biological function in different strains can have an amino acid sequence (and polynucleotide sequence) that is not identical in each of the strains.
  • an immune response directed generally against many allelic variants has been demonstrated.
  • cross-strain antibody binding plus neutralization of infectivity occurs despite amino acid sequence variation of MOMP from strain to strain, indicating that the MOMP, when used as an immunogen, is tolerant of amino acid variations.
  • Polynucleotides encoding homologous polypeptides or allelic variants are retrieved by polymerase chain reaction (PCR) amplification of genomic bacterial DNA extracted by conventional methods. This involves the use of synthetic oligonucleotide primers matching upstream and downstream of the 5′ and 3′ ends of the encoding domain. Suitable primers are designed according to the nucleotide sequence information provided in any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. The procedure is as follows: a primer is selected which consists of 10 to 40, preferably 15 to 25 nucleotides.
  • a standard PCR reaction contains typically 0.5 to 5 Units of Taq DNA polymerase per 100 ⁇ L, 20 to 200 ⁇ M deoxynucleotide each, preferably at equivalent concentrations, 0.5 to 2.5 mM magnesium over the total deoxynucleotide concentration, 10 5 to 10 6 target molecules, and about 20 pmol of each primer. About 25 to 50 PCR cycles are performed, with an annealing temperature 15° C. to 5° C. below the true Tm of the primers.
  • a more stringent annealing temperature improves discrimination against incorrectly annealed primers and reduces incorportion of incorrect nucleotides at the 3′ end of primers.
  • a denaturation temperature of 95° C. to 97° C. is typical, although higher temperatures may be appropriate for dematuration of G+C ⁇ rich targets. The number of cycles performed depends on the starting concentration of target molecules, though typically more than 40 cycles is not recommended as non-specific background products tend to accumulate.
  • An alternative method for retrieving polynucleotides encoding homologous polypeptides or allelic variants is by hybridization screening of a DNA or RNA library. Hybridization procedures are well-known in the art and are described in Ausubel et al., (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994), Silhavy et al. (Silhavy et al. Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, 1984), and Davis et al. (Davis et al. A Manual for Genetic Engineering: Advanced Bacterial Genetics, Cold Spring Harbor Laboratory Press, 1980)).
  • stringent conditions are achieved for both pre-hybridizing and hybridizing incubations (i) within 4-16 hours at 42° C., in 6 ⁇ SSC containing 50% formamide, or (ii) within 4-16 hours at 65° C. in an aqueous 6 ⁇ SSC solution (1 M NaCl, 0.1 M sodium citrate (pH 7.0)).
  • hybridization experiments are performed at a temperature from 60 to 68° C., e.g. 65° C.
  • stringent hybridization conditions can be achieved in 6 ⁇ SSC, preferably in 2 ⁇ SSC or 1 ⁇ SSC, more preferably in 0.5 ⁇ SSc, 0.3 ⁇ SSC or 0.1 ⁇ SSC (in the absence of formamide).
  • 1 ⁇ SSC contains 0.15 M NaCl and 0.015 M sodium citrate.
  • Useful homologs and fragments thereof that do not occur naturally are designed using known methods for identifying regions of an antigen that are likely to tolerate amino acid sequence changes and/or deletions. As an example, homologous polypeptides from different species are compared; conserved sequences are identified. The more divergent sequences are the most likely to tolerate sequence changes. Homology among sequences may be analyzed using, as an example, the BLAST homology searching algorithm of Altschul et al., Nucleic Acids Res.; 25:3389-3402 (1997).
  • sequences are modified such that they become more reactive to T- and/or B-cells, based on computer-assisted analysis of probable T- or B-cell epitopes
  • Yet another alternative is to mutate a particular amino acid residue or sequence within the polypeptide in vitro, then screen the mutant polypeptides for their ability to prevent or treat Chlamydia infection according to the method outlined below.
  • conferring protection is meant that there is a reduction in severity of any of the effects of Chlamydia infection, in comparison with a control animal which was not immunized with the test homolog or fragment.
  • polypeptide derivatives are provided that are partial sequences of any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, partial sequences of polypeptide sequences homologous to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, polypeptides derived from full-length polypeptides by internal deletion, and fusion proteins.
  • polypeptide fragments preferably are at least 12 amino acids in length.
  • polypeptide fragments are at least 15 amino acids, preferably at least 20, 25, 30, 35, 40, 45, 50 amino acids, more preferably at least 55, 60, 65, 70, 75 amino acids, and most preferably at least 80, 85, 90, 95, 100 amino acids in length.
  • Polynucleotides of 30 to 600 nucleotides encoding partial sequences of sequences homologous to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 are retrieved by PCR amplification using the parameters outlined above and using primers matching the sequences upstream and downstream of the 5′ and 3′ ends of the fragment to be amplified.
  • the template polynucleotide for such amplification is either the full length polynucleotide homologous to any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, or a polynucleotide contained in a mixture of polynucleotides such as a DNA or RNA library.
  • Short peptides that are fragments of any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 or its homologous sequences, are obtained directly by chemical synthesis (E. Gross and H. J. Meinhofer, 4 The Peptides: Analysis, Synthesis, Biology; Modern Techniques of Peptide Synthesis, John Wiley & Sons (1981), and M. Bodanzki, Principles of Peptide Synthesis, Springer-Verlag (1984)).
  • polypeptide derivatives are designed using computer-assisted analysis of amino acid sequences. This would identify probable surface-exposed, antigenic regions (Hughes et al., 1992. Infect. Immun. 60(9):3497). Analysis of 6 amino acid sequences contained in any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, based on the product of flexibility and hydrophobicity propensities using the program SEQSEE (Wishart D S, et al. “SEQSEE: a comprehensive program suite for protein sequence analysis.” Comput Appl Biosci .
  • Epitopes which induce a protective T cell-dependent immune response are present throughout the length of the polypeptide. However, some epitopes may be masked by secondary and tertiary structures of the polypeptide. To reveal such masked epitopes large internal deletions are created which remove much of the original protein structure and exposes the masked epitopes. Such internal deletions sometimes effect the additional advantage of removing immunodominant regions of high variability among strains.
  • Polynucleotides encoding polypeptide fragments and polypeptides having large internal deletions are constructed using standard methods (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994). Such methods include standard PCR, inverse PCR, restriction enzyme treatment of cloned DNA molecules, or the method of Kunkel et al. (Kunkel et al. Proc. Natl. Acad. Sci. USA (1985) 82:448). Components for these methods and instructions for their use are readily available from various commercial sources such as Stratagene. Once the deletion mutants have been constructed, they are tested for their ability to prevent or treat Chlamydia infection as described above.
  • a fusion polypeptide is one that contains a polypeptide or a polypeptide derivative of the invention fused at the N- or C-terminal end to any other polypeptide (hereinafter referred to as a peptide tail).
  • a simple way to obtain such a fusion polypeptide is by translation of an in-frame fusion of the polynucleotide sequences, i.e., a hybrid gene.
  • the hybrid gene encoding the fusion polypeptide is inserted into an expression vector which is used to transform or transfect a host cell.
  • polynucleotide sequence encoding the polypeptide or polypeptide derivative is inserted into an expression vector in which the polynucleotide encoding the peptide tail is already present.
  • vectors and instructions for their use are commercially available, e.g. the pMal-c2 or pMal-p2 system from New England Biolabs, in which the peptide tail is a maltose binding protein, the glutathione-S-transferase system of Pharmacia, or the His-Tag system available from Novagen.
  • a fusion polypeptide is one where the polypeptide or homolog or fragment of the invention is fused to a polypeptide having adjuvant activity, such as subunit B of either cholera toxin or E. coli heat-labile toxin.
  • a polypeptide having adjuvant activity such as subunit B of either cholera toxin or E. coli heat-labile toxin.
  • Another advantageous fusion is one where the polypeptide, homolog or fragment is fused to a strong T-cell epitope or B-cell epitope.
  • Such an epitope may be one known in the art (e.g. the Hepatitis B virus core antigen, D. R. Millich et al., “Antibody production to the nucleocapsid and envelope of the Hepatitis B virus primed by a single synthetic T cell site”, Nature. 1987.
  • a fusion polypeptide comprising T- or B-cell epitopes from any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 or its homolog or fragment, wherein the epitopes are derived from multiple variants of said polypeptide or homolog or fragment, each variant differing from another in the location and sequence of its epitope within the polypeptide.
  • Such a fusion is effective in the prevention and treatment of Chlamydia infection since it optimizes the T- and B-cell response to the overall polypeptide, homolog or fragment.
  • the polypeptide of the invention is fused to the N-, or preferably, to the C-terminal end of the polypeptide having adjuvant activity or T- or B-cell epitope.
  • a polypeptide fragment of the invention is inserted internally within the amino acid sequence of the polypeptide having adjuvant activity.
  • the T- or B-cell epitope may also be inserted internally within the amino acid sequence of the polypeptide of the invention.
  • polynucleotides of the invention also encode hybrid-precursor polypeptides containing heterologous signal peptides, which mature into polypeptides of the invention.
  • heterologous signal peptide is meant a signal peptide that is not found in naturally-occurring precursors of polypeptides of the invention.
  • Polynucleotide molecules according to the invention have various applications.
  • a DNA molecule is used, for example, (i) in a process for producing the encoded polypeptide in a recombinant host system, (ii) in the construction of vaccine vectors such as poxviruses, which are further used in methods and compositions for preventing and/or treating Chlamydia infection, (iii) as a vaccine agent (as well as an RNA molecule), in a naked form or formulated with a delivery vehicle and, (iv) in the construction of attenuated Chlamydia strains that can over-express a polynucleotide of the invention or express it in a non-toxic, mutated form.
  • Selected genes from pathogenic micro-organisms within an eukaryotic expression plasmid are useful as vaccines.
  • Expression plasmids contain methylated CpG motifs that elicit innate cytokine responses that promote the canalization of CD4 T cell responses to a Thl cytokine secretion pattern.
  • the intracellular synthesis of the microbial protein especially within transfected professional antigen-presenting cells, facilitates the presentation of antigen on class I and class II molecules and the induction of cell-mediated immunity.
  • the use of one or a number of microbial protein-coding genes allows the presentation of protective antigens to the immune system to occur in the absence of microbe-directed immune evasion mechanisms and in the absence of competing or pathologic antigens.
  • Immune responses primed by DNA vaccines are also readily amplified by protein-antigen immunization. Thus, immunization with DNA vaccines is particularly relevant to chlamydial vaccine design.
  • a second aspect of the invention encompasses (i) an expression cassette containing a DNA molecule of the invention placed under the control of the elements required for expression, in particular under the control of an appropriate promoter; (ii) an expression vector containing an expression cassette of the invention; (iii) a procaryotic or eucaryotic cell transformed or transfected with an expression cassette and/or vector of the invention, as well as (iv) a process for producing a polypeptide or polypeptide derivative encoded by a polynucleotide of the invention, which involves culturing a procaryotic or eucaryotic cell transformed or transfected with an expression cassette and/or vector of the invention, under conditions that allow expression of the DNA molecule of the invention and, recovering the encoded polypeptide or polypeptide derivative from the cell culture.
  • a recombinant expression system is selected from procaryotic and eucaryotic hosts.
  • Eucaryotic hosts include yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris ), mammalian cells (e.g., COS1, NIH3T3, or JEG3 cells), arthropods cells (e.g., Spodoptera frugiperda (SF9) cells), and plant cells.
  • a preferred expression system is a procaryotic host such as E. coli .
  • Bacterial and eucaryotic cells are available from a number of different sources including commercial sources to those skilled in the art, e.g., the American Type Culture Collection (ATCC; Rockville, Md.). Commercial sources of cells used for recombinant protein expression also provide instructions for usage of the cells.
  • the choice of the expression system depends on the features desired for the expressed polypeptide. For example, it may be useful to produce a polypeptide of the invention in a particular lipidated form or any other form.
  • the host In selecting a vector, the host must be chosen that is compatible with the vector which is to exist and possibly replicate in it. Considerations are made with respect to the vector copy number, the ability to control the copy number, expression of other proteins such as antibiotic resistance.
  • an expression control sequence a number of variables are considered. Among the important variable are the relative strength of the sequence (e.g. the ability to drive expression under various conditions), the ability to control the sequence's function, compatibility between the polynucleotide to be expressed and the control sequence (e.g. secondary structures are considered to avoid hairpin structures which prevent efficient transcription).
  • unicellular hosts are selected which are compatible with the selected vector, tolerant of any possible toxic effects of the expressed product, able to secrete the expressed product efficiently if such is desired, to be able to express the product in the desired conformation, to be easily scaled up, and to which ease of purification of the final product.
  • an expression cassette includes a promoter that is functional in the selected host system and can be constitutive or inducible; a ribosome binding site; a start codon (ATG) if necessary; a region encoding a signal peptide, e.g., a lipidation signal peptide; a DNA molecule of the invention; a stop codon; and optionally a 3′ terminal region (translation and/or transcription terminator).
  • the signal peptide encoding region is adjacent to the polynucleotide of the invention and placed in proper reading frame.
  • the signal peptide-encoding region is homologous or heterologous to the DNA molecule encoding the mature polypeptide and is compatible with the secretion apparatus of the host used for expression.
  • the open reading frame constituted by the DNA molecule of the invention, solely or together with the signal peptide, is placed under the control of the promoter so that transcription and translation occur in the host system.
  • Promoters and signal peptide encoding regions are widely known and available to those skilled in the art and include, for example, the promoter of Salmonella typhimurium (and derivatives) that is inducible by arabinose (promoter araB) and is functional in Gram-negative bacteria such as E. coli (as described in U.S. Pat. No.
  • the expression cassette is typically part of an expression vector, which is selected for its ability to replicate in the chosen expression system.
  • Expression vectors e.g., plasmids or viral vectors
  • plasmids or viral vectors can be chosen, for example, from those described in Pouwels et al. (Cloning Vectors: A Laboratory Manual 1985, Supp. 1987). Suitable expression vectors can be purchased from various commercial sources.
  • a recombinant polypeptide of the invention (or a polypeptide derivative) is produced and remains in the intracellular compartment, is secreted/excreted in the extracellular medium or in the periplasmic space, or is embedded in the cellular membrane.
  • the polypeptide is recovered in a substantially purified form from the cell extract or from the supernatant after centrifugation of the recombinant cell culture.
  • the recombinant polypeptide is purified by antibody-based affinity purification or by other well-known methods that can be readily adapted by a person skilled in the art, such as fusion of the polynucleotide encoding the polypeptide or its derivative to a small affinity binding domain.
  • Antibodies useful for purifying by immunoaffinity the polypeptides of the invention are obtained as described below.
  • a polynucleotide of the invention can also be useful as a vaccine. There are two major routes, either using a viral or bacterial host as gene delivery vehicle (live vaccine vector) or administering the gene in a free form, e.g., inserted into a plasmid. Therapeutic or prophylactic efficacy of a polynucleotide of the invention is evaluated as described below.
  • a third aspect of the invention provides (i) a vaccine vector such as a poxvirus, containing a DNA molecule of the invention, placed under the control of elements required for expression; (ii) a composition of matter comprising a vaccine vector of the invention, together with a diluent or carrier; specifically (iii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a vaccine vector of the invention; (iv) a method for inducing an immune response against Chlamydia in a mammal (e.g., a human; alternatively, the method can be used in veterinary applications for treating or preventing Chlamydia infection of animals, e.g., cats or birds), which involves administering to the mammal an immunogenically effective amount of a vaccine vector of the invention to elicit a protective or therapeutic immune response to Chlamydia ; and particularly, (v) a method for preventing and/or treating a Chlamydia
  • the third aspect of the invention encompasses the use of a vaccine vector of the invention in the preparation of a medicament for preventing and/or treating Chlamydia infection.
  • a vaccine vector expresses one or several polypeptides or derivatives of the invention.
  • the vaccine vector may express additionally a cytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12), that enhances the immune response (adjuvant effect).
  • IL-2 interleukin-2
  • IL-12 interleukin-12
  • composition comprising several vaccine vectors, each of them capable of expressing a polypeptide or derivative of the invention.
  • a composition may also comprise a vaccine vector capable of expressing an additional Chlamydia antigen, or a subunit, fragment, homolog, mutant, or derivative thereof; optionally together with or a cytokine such as IL-2 or IL-12.
  • a general principle is that recognition of a particular antigen is not in itself sufficient to produce an effective immune response. In some cases, a cell-mediated response is appropriate; in others, antibody.
  • Antigens of microorganisms vary considerably in their accessibility to cells of the immune system. Antigens which normally occur inside a pathogen may become accessible only when the pathogen or an infected cell is killed. Even antigens expressed at the cell surface may present only a limited range of their potential epitopes for antibody binding, depending on their orientation in the membrane. Protective structures, such as bacterial capsules, further limit the effective recognition of epitopes.
  • the primary effectors against extracellular pathogens are antibody and complement. Binding of antibody to receptors on the pathogen can prevent it from attaching to its target cell. Antibody alone, or more effectively in association with complement, opsonizes pathogens for uptake by phagocytes expressing Fc receptors and complement receptors CR1 and CR3. Usually this will lead to intracellular destruction of the pathogen but if the phagocyte is unable to destroy it and is a facultative host cell, then antibody may actually promote the spread of infection. Such an eventuality, however, depends on the dynamic balance between the actions of the humoral and cell-mediated immune responses.
  • Tables 1, 3, 4, 6, 8, 10, 12, 14, 16 and 18, as well as corresponding FIGS. 31 to 40 demonstrate that the polypeptides disclosed herein are immunogenic. Furthermore, these Figures demonstrate that the polypeptides disclosed herein confer immunoprotection from Chlamydia infection, as evidenced by accelerated clearance of pulmonary infection. Such reduction in the severity of effects of Chlamydia infection is evidence that the polypeptides have generated an active functional immune response against the pathogen, rather than a mere antibody response against the antigen.
  • C. trachomatis mouse pneumonitis is the most widely tested biovar among the three C. trachomatis biovars (trachoma, lymphogranuloma venereum, and MoPn).
  • human biovars have also been used in animal models, they normally require high inocula or pretreatment with progesterone.
  • MoPn which was originally isolated from mouse tissues, is thought to be a natural murine pathogen and thus offers an evolutionarily adapted pathogen for analysis of host-pathogen interactions.
  • Vaccination methods for treating or preventing infection in a mammal comprises use of a vaccine vector of the invention to be administered by any conventional route, particularly to a mucosal (e.g., ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract) surface or via the parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route.
  • Preferred routes depend upon the choice of the vaccine vector. Treatment may be effected in a single dose or repeated at intervals. The appropriate dosage depends on various parameters understood by skilled artisans such as the vaccine vector itself, the route of administration or the condition of the mammal to be vaccinated (weight, age and the like).
  • Live vaccine vectors available in the art include viral vectors such as adenoviruses and poxviruses as well as bacterial vectors, e.g., Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bilié de Calmette - Guérin (BCG), and Streptococcus.
  • viral vectors such as adenoviruses and poxviruses
  • bacterial vectors e.g., Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bilié de Calmette - Guérin (BCG), and Streptococcus.
  • adenovirus vector An example of an adenovirus vector, as well as a method for constructing an adenovirus vector capable of expressing a DNA molecule of the invention, are described in U.S. Pat. No. 4,920,209.
  • Poxvirus vectors include vaccinia and canary pox virus, described in U.S. Pat. No. 4,722,848 and U.S. Pat. No. 5,364,773, respectively.
  • Poxvirus vectors capable of expressing a polynucleotide of the invention are obtained by homologous recombination as described in Kieny et al., Nature (1984) 312:163 so that the polynucleotide of the invention is inserted in the viral genome under appropriate conditions for expression in mammalian cells.
  • the dose of vaccine viral vector for therapeutic or prophylactic use, can be of from about 1 ⁇ 10 4 to about 1 ⁇ 10 11 , advantageously from about 1 ⁇ 10 7 to about 1 ⁇ 10 10 , preferably of from about 1 ⁇ 10 7 to about 1 ⁇ 10 9 plaque-forming units per kilogram.
  • viral vectors are administered parenterally; for example, in 3 doses, 4 weeks apart. It is preferable to avoid adding a chemical adjuvant to a composition containing a viral vector of the invention and thereby minimizing the immune response to the viral vector itself.
  • Non-toxicogenic Vibrio cholerae mutant strains that are useful as a live oral vaccine are known.
  • Mekalanos et al., Nature (1983) 306:551 and U.S. Pat. No. 4,882,278 describe strains which have a substantial amount of the coding sequence of each of the two ctxA alleles deleted so that no functional cholerae toxin is produced.
  • WO 92/11354 describes a strain in which the irgA locus is inactivated by mutation; this mutation can be combined in a single strain with ctxA mutations.
  • WO 94/01533 describes a deletion mutant lacking functional ctxA and attRS1 DNA sequences.
  • An effective vaccine dose of a Vibrio cholerae strain capable of expressing a polypeptide or polypeptide derivative encoded by a DNA molecule of the invention contains about 1 ⁇ 10 5 to about 1 ⁇ 10 9 , preferably about 1 ⁇ 10 6 to about 1 ⁇ 10 8 , viable bacteria in a volume appropriate for the selected route of administration.
  • Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.
  • Attenuated Salmonella typhimurium strains genetically engineered for recombinant expression of heterologous antigens or not, and their use as oral vaccines are described in Nakayama et al. (Bio/Technology (1988) 6:693) and WO 92/11361.
  • Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.
  • the polynucleotide of the invention is inserted into the bacterial genome or remains in a free state as part of a plasmid.
  • composition comprising a vaccine bacterial vector of the present invention may further contain an adjuvant.
  • adjuvants are known to those skilled in the art. Preferred adjuvants are selected as provided below.
  • a fourth aspect of the invention provides (i) a composition of matter comprising a polynucleotide of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a polynucleotide of the invention; (iii) a method for inducing an immune response against Chlamydia in a mammal by administration of an immunogenically effective amount of a polynucleotide of the invention to elicit a protective immune response to Chlamydia ; and particularly, (iv) a method for preventing and/or treating a Chlamydia (e.g., C.
  • a Chlamydia e.g., C.
  • the fourth aspect of the invention encompasses the use of a polynucleotide of the invention in the preparation of a medicament for preventing and/or treating Chlamydia infection.
  • a preferred use includes the use of a DNA molecule placed under conditions for expression in a mammalian cell, especially in a plasmid that is unable to replicate in mammalian cells and to substantially integrate in a mammalian genome.
  • polynucleotides of the invention include their administration to a mammal as a vaccine, for therapeutic or prophylactic purposes.
  • Such polynucleotides are used in the form of DNA as part of a plasmid that is unable to replicate in a mammalian cell and unable to integrate into the mammalian genome.
  • a DNA molecule is placed under the control of a promoter suitable for expression in a mammalian cell.
  • the promoter functions either ubiquitously or tissue-specifically. Examples of non-tissue specific promoters include the early Cytomegalovirus (CMV) promoter (described in U.S. Pat. No.
  • CMV Cytomegalovirus
  • tissue-specific promoter is the desmin promoter which drives expression in muscle cells (Li et al., Gene (1989) 78:243, Li & Paulin, J. Biol. Chem. (1991) 266:6562 and Li & Paulin, J. Biol. Chem. (1993) 268:10403).
  • Use of promoters is well-known to those skilled in the art. Useful vectors are described in numerous publications, specifically WO 94/21797 and Hartikka et al., Human Gene Therapy (1996) 7:1205.
  • Polynucleotides of the invention which are used as vaccines encode either a precursor or a mature form of the corresponding polypeptide.
  • the signal peptide is either homologous or heterologous.
  • a eucaryotic leader sequence such as the leader sequence of the tissue-type plasminogen factor (tPA) is preferred.
  • a composition of the invention contains one or several polynucleotides with optionally at least one additional polynucleotide encoding another Chlamydia antigen such as urease subunit A, B, or both, or a fragment, derivative, mutant, or analog thereof.
  • the composition may also contain an additional polynucleotide encoding a cytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12) so that the immune response is enhanced.
  • IL-2 interleukin-2
  • IL-12 interleukin-12
  • Standard techniques of molecular biology for preparing and purifying polynucleotides are used in the preparation of polynucleotide therapeutics of the invention.
  • a polynucleotide of the invention is formulated according to various methods outlined below.
  • One method utililizes the polynucleotide in a naked form, free of any delivery vehicles.
  • a polynucleotide is simply diluted in a physiologically acceptable solution such as sterile saline or sterile buffered saline, with or without a carrier.
  • the carrier preferably is isotonic, hypotonic, or weakly hypertonic, and has a relatively low ionic strength, such as provided by a sucrose solution, e.g., a solution containing 20% sucrose.
  • An alternative method utilizes the polynucleotide in association with agents that assist in cellular uptake.
  • agents that assist in cellular uptake include (i) chemicals that modify cellular permeability, such as bupivacaine (see, e.g., WO 94/16737), (ii) liposomes for encapsulation of the polynucleotide, or (iii) cationic lipids or silica, gold, or tungsten microparticles which associate themselves with the polynucleotides.
  • Cationic lipids are also known in the art and are commonly used for gene delivery.
  • Such lipids include LipofectinTM also known as DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (1,2-bis(oleyloxy)-3-(trimethylammonio)propane), DDAB (dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycyl spermine) and cholesterol derivatives such as DC-Chol (3 beta-(N-(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol).
  • DC-Chol beta-(N-(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol
  • Cationic lipids for gene delivery are preferably used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine), as described in WO 90/11092 as an example.
  • DOPE dioleyl phosphatidylethanolamine
  • Formulation containing cationic liposomes may optionally contain other transfection-facilitating compounds.
  • transfection-facilitating compounds A number of them are described in WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/02397. They include spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane (see, for example, WO 93/18759) and membrane-permeabilizing compounds such as GALA, Gramicidine S, and cationic bile salts (see, for example, WO 93/19768).
  • Gold or tungsten microparticles are used for gene delivery, as described in WO 91/00359, WO 93/17706, and Tang et al. Nature (1992) 356:152.
  • the microparticle-coated polynucleotide is injected via intradermal or intraepidermal routes using a needleless injection device (“gene gun”), such as those described in U.S. Pat. No. 4,945,050, U.S. Pat. No. 5,015,580, and WO 94/24263.
  • the amount of DNA to be used in a vaccine recipient depends, e.g., on the strength of the promoter used in the DNA construct, the immunogenicity of the expressed gene product, the condition of the mammal intended for administration (e.g., the weight, age, and general health of the mammal), the mode of administration, and the type of formulation.
  • a therapeutically or prophylactically effective dose from about 1 ⁇ g to about 1 mg, preferably, from about 10 ⁇ g to about 800 ⁇ g and, more preferably, from about 25 ⁇ g to about 250 ⁇ g, can be administered to human adults.
  • the administration can be achieved in a single dose or repeated at intervals.
  • the route of administration is any conventional route used in the vaccine field.
  • a polynucleotide of the invention is administered via a mucosal surface, e.g., an ocular, intranasal, pulmonary, oral, intestinal, rectal, vaginal, and urinary tract surface; or via a parenteral route, e.g., by an intravenous, subcutaneous, intraperitoneal, intradermal, intraepidermal, or intramuscular route.
  • a polynucleotide formulated in association with bupivacaine is advantageously administered into muscles.
  • the formulation can be advantageously injected via intravenous, intranasal (aerosolization), intramuscular, intradermal, and subcutaneous routes.
  • a polynucleotide in a naked form can advantageously be administered via the intramuscular, intradermal, or sub-cutaneous routes.
  • such a composition can also contain an adjuvant. If so, a systemic adjuvant that does not require concomitant administration in order to exhibit an adjuvant effect is preferable such as, e.g., QS21, which is described in U.S. Pat. No. 5,057,546.
  • a fifth aspect of the invention provides a nucleotide probe or primer having a sequence found in or derived by degeneracy of the genetic code from a sequence shown in any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19.
  • probe refers to DNA (preferably single stranded) or RNA molecules (or modifications or combinations thereof) that hybridize under the stringent conditions, as defined above, to nucleic acid molecules having any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 or to a sequence homologous to any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, or to its complementary or anti-sense sequence.
  • probes are significantly shorter than full-length sequences. Such probes contain from about 5 to about 100, preferably from about 10 to about 80, nucleotides.
  • probes have sequences that are at least 75%, preferably at least 80% or 85%, more preferably 90% or 95% homologous to a portion of any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 or that are complementary to such sequences.
  • Probes may contain modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, or diamino-2,6-purine. Sugar or phosphate residues may also be modified or substituted.
  • a deoxyribose residue may be replaced by a polyamide (Nielsen et al., Science (1991) 254:1497) and phosphate residues may be replaced by ester groups such as diphosphate, alkyl, arylphosphonate and phosphorothioate esters.
  • ester groups such as diphosphate, alkyl, arylphosphonate and phosphorothioate esters.
  • 2′-hydroxyl group on ribonucleotides may be modified by including such groups as alkyl groups.
  • Probes of the invention are used in diagnostic tests, as capture or detection probes. Such capture probes are conventionally immobilized on a solid support, directly or indirectly, by covalent means or by passive adsorption.
  • a detection probe is labelled by a detection marker selected from: radioactive isotopes, enzymes such as peroxidase, alkaline phosphatase, and enzymes able to hydrolyze a chromogenic, fluorogenic, or luminescent substrate, compounds that are chromogenic, fluorogenic, or luminescent, nucleotide base analogs, and biotin.
  • Probes of the invention are used in any conventional hybridization technique, such as dot blot (Maniatis et al., Molecular Cloning: A Laboratory Manual (1982) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), Southern blot (Southern, J. Mol. Biol. (1975) 98:503), northern blot (identical to Southern blot with the exception that RNA is used as a target), or the sandwich technique (Dunn et al., Cell (1977) 12:23).
  • dot blot Maniatis et al., Molecular Cloning: A Laboratory Manual (1982) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  • Southern blot Southern blot
  • northern blot identical to Southern blot with the exception that RNA is used as a target
  • sandwich technique Nordstrom et al., Cell (1977) 12:23).
  • the latter technique involves the use of a specific capture probe and/or a specific detection probe with nucleo
  • a primer is a probe of usually about 10 to about 40 nucleotides that is used to initiate enzymatic polymerization of DNA in an amplification process (e.g., PCR), in an elongation process, or in a reverse transcription method. Primers used in diagnostic methods involving PCR are labeled by methods known in the art.
  • the invention also encompasses (i) a reagent comprising a probe of the invention for detecting and/or identifying the presence of Chlamydia in a biological material; (ii) a method for detecting and/or identifying the presence of Chlamydia in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA or RNA is extracted from the material and denatured, and (c) exposed to a probe of the invention, for example, a capture, detection probe or both, under stringent hybridization conditions, such that hybridization is detected; and (iii) a method for detecting and/or identifying the presence of Chlamydia in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA is extracted therefrom, (c) the extracted DNA is primed with at least one, and preferably two, primers of the invention and amplified by polymerase chain reaction, and (d
  • a sixth aspect of the invention features a substantially purified polypeptide or polypeptide derivative having an amino acid sequence encoded by a polynucleotide of the invention.
  • a “substantially purified polypeptide” as used herein is defined as a polypeptide that is separated from the environment in which it naturally occurs and/or that is free of the majority of the polypeptides that are present in the environment in which it was synthesized.
  • a substantially purified polypeptide is free from cytoplasmic polypeptides.
  • the polypeptides of the invention may be purified from a natural source, i.e., a Chlamydia strain, or produced by recombinant means.
  • Consistent with the sixth aspect of the invention are polypeptides, homologs or fragments which are modified or treated to enhance their immunogenicity in the target animal, in whom the polypeptide, homolog or fragments are intended to confer protection against Chlamydia .
  • modifications or treatments include: amino acid substitutions with an amino acid derivative such as 3-methyhistidine, 4-hydroxyproline, 5-hydroxylysine etc., modifications or deletions which are carried out after preparation of the polypeptide, homolog or fragment, such as the modification of free amino, carboxyl or hydroxyl side groups of the amino acids.
  • a monospecific hyperimmune antiserum is raised against a purified reference polypeptide, a fusion polypeptide (for example, an expression product of MBP, GST, or His-tag systems, the description and instructions for use of which are contained in Invitrogen product manuals for pcDNA3.1/Myc-His(+) A, B, and C and for the XpressTM System Protein Purification), or a synthetic peptide predicted to be antigenic.
  • a fusion polypeptide for example, an expression product of MBP, GST, or His-tag systems, the description and instructions for use of which are contained in Invitrogen product manuals for pcDNA3.1/Myc-His(+) A, B, and C and for the XpressTM System Protein Purification
  • a synthetic peptide predicted to be antigenic where an antiserum is raised against a fusion polypeptide, two different fusion systems are employed. Specific antigenicity can be determined according to a number of methods, including Western blot (Towbin
  • the product to be screened is submitted to SDS-Page electrophoresis as described by Laemmli (Nature (1970) 227:680).
  • the material is further incubated with the monospecific hyperimmune antiserum diluted in the range of dilutions from about 1:5 to about 1:5000, preferably from about 1:100 to about 1:500.
  • Specific antigenicity is shown once a band corresponding to the product exhibits reactivity at any of the dilutions in the above range.
  • the product to be screened is preferably used as the coating antigen.
  • a purified preparation is preferred, although a whole cell extract can also be used. Briefly, about 100 ⁇ l of a preparation at about 10 ⁇ g protein/ml are distributed into wells of a 96-well polycarbonate ELISA plate. The plate is incubated for 2 hours at 37° C. then overnight at 4° C. The plate is washed with phosphate buffer saline (PBS) containing 0.05% Tween 20 (PBS/Tween buffer). The wells are saturated with 250 ⁇ l PBS containing 1% bovine serum albumin (BSA) to prevent non-specific antibody binding.
  • PBS phosphate buffer saline
  • BSA bovine serum albumin
  • the plate After 1 hour incubation at 37° C., the plate is washed with PBS/Tween buffer. The antiserum is serially diluted in PBS/Tween buffer containing 0.5% BSA. 100 ⁇ l of dilutions are added per well. The plate is incubated for 90 minutes at 37° C., washed and evaluated according to standard procedures. For example, a goat anti-rabbit peroxidase conjugate is added to the wells when specific antibodies were raised in rabbits. Incubation is carried out for 90 minutes at 37° C. and the plate is washed. The reaction is developed with the appropriate substrate and the reaction is measured by colorimetry (absorbance measured spectrophotometrically). Under the above experimental conditions, a positive reaction is shown by O.D. values greater than a non immune control serum.
  • a purified product is preferred, although a whole cell extract can also be used.
  • a solution of the product at about 100 ⁇ g/ml is serially two-fold diluted in 50 mM Tris-HCl (pH 7.5). 100 ⁇ l of each dilution are applied to a nitrocellulose membrane 0.45 ⁇ m set in a 96-well dot blot apparatus (Biorad). The buffer is removed by applying vacuum to the system. Wells are washed by addition of 50 mM Tris-HCl (pH 7.5) and the membrane is air-dried.
  • the membrane is saturated in blocking buffer (50 mM Tris-HCl (pH 7.5) 0.15 M NaCl, 10 g/L skim milk) and incubated with an antiserum dilution from about 1:50 to about 1:5000, preferably about 1:500.
  • the reaction is revealed according to standard procedures. For example, a goat anti-rabbit peroxidase conjugate is added to the wells when rabbit antibodies are used. Incubation is carried out 90 minutes at 37° C. and the blot is washed. The reaction is developed with the appropriate substrate and stopped. The reaction is measured visually by the appearance of a colored spot, e.g., by colorimetry. Under the above experimental conditions, a positive reaction is shown once a colored spot is associated with a dilution of at least about 1:5, preferably of at least about 1:500.
  • a seventh aspect of the invention provides (i) a composition of matter comprising a polypeptide of the invention together with a diluent or carrier; specifically (ii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a polypeptide of the invention; (iii) a method for inducing an immune response against Chlamydia in a mammal, by administering to the mammal an immunogenically effective amount of a polypeptide of the invention to elicit a protective immune response to Chlamydia ; and particularly, (iv) a method for preventing and/or treating a Chlamydia (e.g., C.
  • a Chlamydia e.g., C.
  • the seventh aspect of the invention encompasses the use of a polypeptide of the invention in the preparation of a medicament for preventing and/or treating Chlamydia infection.
  • the immunogenic compositions of the invention are administered by conventional routes known the vaccine field, in particular to a mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface or via the parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route.
  • a mucosal e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract
  • parenteral e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal
  • the choice of administration route depends upon a number of parameters, such as the adjuvant associated with the polypeptide. If a mucosal adjuvant is used, the intranasal or oral route is preferred.
  • lipid formulation or an aluminum compound is used, the parenteral route is preferred with the sub-cutaneous or intramuscular route being most preferred.
  • the choice also depends upon the nature of the vaccine agent. For example, a polypeptide of the invention fused to CTB or LTB is best administered to a mucosal surface.
  • composition of the invention contains one or several polypeptides or derivatives of the invention.
  • the composition optionally contains at least one additional Chlamydia antigen, or a subunit, fragment, homolog, mutant, or derivative thereof.
  • a polypeptide or derivative thereof is formulated into or with liposomes, preferably neutral or anionic liposomes, microspheres, ISCOMS, or virus-like-particles (VLPs) to facilitate delivery and/or enhance the immune response.
  • liposomes preferably neutral or anionic liposomes, microspheres, ISCOMS, or virus-like-particles (VLPs) to facilitate delivery and/or enhance the immune response.
  • Adjuvants other than liposomes and the like are also used and are known in the art. Adjuvants may protect the antigen from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. An appropriate selection can conventionally be made by those skilled in the art, for example, from those described below (under the eleventh aspect of the invention).
  • Treatment is achieved in a single dose or repeated as necessary at intervals, as can be determined readily by one skilled in the art. For example, a priming dose is followed by three booster doses at weekly or monthly intervals.
  • An appropriate dose depends on various parameters including the recipient (e.g., adult or infant), the particular vaccine antigen, the route and frequency of administration, the presence/absence or type of adjuvant, and the desired effect (e.g., protection and/or treatment), as can be determined by one skilled in the art.
  • a vaccine antigen of the invention is administered by a mucosal route in an amount from about 10 ⁇ g to about 500 mg, preferably from about 1 mg to about 200 mg.
  • the dose usually does not exceed about 1 mg, preferably about 100 ⁇ g.
  • polynucleotides and polypeptides of the invention may be used sequentially as part of a multistep immunization process.
  • a mammal is initially primed with a vaccine vector of the invention such as a pox virus, e.g., via the parenteral route, and then boosted twice with the polypeptide encoded by the vaccine vector, e.g., via the mucosal route.
  • liposomes associated with a polypeptide or derivative of the invention is also used for priming, with boosting being carried out mucosally using a soluble polypeptide or derivative of the invention in combination with a mucosal adjuvant (e.g., LT).
  • a mucosal adjuvant e.g., LT
  • a polypeptide derivative of the invention is also used in accordance with the seventh aspect as a diagnostic reagent for detecting the presence of anti- Chlamydia antibodies, e.g., in a blood sample.
  • Such polypeptides are about 5 to about 80, preferably about 10 to about 50 amino acids in length. They are either labeled or unlabeled, depending upon the diagnostic method. Diagnostic methods involving such a reagent are described below.
  • a polypeptide or polypeptide derivative is produced and purified using known laboratory techniques.
  • the polypeptide or polypeptide derivative may be produced as a fusion protein containing a fused tail that facilitates purification.
  • the fusion product is used to immunize a small mammal, e.g., a mouse or a rabbit, in order to raise antibodies against the polypeptide or polypeptide derivative (monospecific antibodies).
  • an eighth aspect of the invention provides a monospecific antibody that binds to a polypeptide or polypeptide derivative of the invention.
  • monospecific antibody an antibody that is capable of reacting with a unique naturally-occurring Chlamydia polypeptide.
  • An antibody of the invention is either polyclonal or monoclonal.
  • Monospecific antibodies may be recombinant, e.g., chimeric (e.g., constituted by a variable region of murine origin associated with a human constant region), humanized (a human immunoglobulin constant backbone together with hypervariable region of animal, e.g., murine, origin), and/or single chain. Both polyclonal and monospecific antibodies may also be in the form of immunoglobulin fragments, e.g., F(ab)′2 or Fab fragments.
  • the antibodies of the invention are of any isotype, e.g., IgG or IgA, and polyclonal antibodies are of a single isotype or a mixture of isotypes.
  • Antibodies against the polypeptides, homologs or fragments of the present invention are generated by immunization of a mammal with a composition comprising said polypeptide, homolog or fragment. Such antibodies may be polyclonal or monoclonal. Methods to produce polyclonal or monoclonal antibodies are well known in the art. For a review, see “Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Eds. E. Harlow and D. Lane (1988), and D. E. Yelton et al., 1981. Ann. Rev. Biochem. 50:657-680. For monoclonal antibodies, see Kohler & Milstein (1975) Nature 256:495-497.
  • the antibodies of the invention which are raised to a polypeptide or polypeptide derivative of the invention, are produced and identified using standard immunological assays, e.g., Western blot analysis, dot blot assay, or ELISA (see, e.g., Coligan et al., Current Protocols in Immunology (1994) John Wiley & Sons, Inc., New York, N.Y.).
  • the antibodies are used in diagnostic methods to detect the presence of a Chlamydia antigen in a sample, such as a biological sample.
  • the antibodies are also used in affinity chromatography for purifying a polypeptide or polypeptide derivative of the invention. As is discussed further below, such antibodies may be used in prophylactic and therapeutic passive immunization methods.
  • a ninth aspect of the invention provides (i) a reagent for detecting the presence of Chlamydia in a biological sample that contains an antibody, polypeptide, or polypeptide derivative of the invention; and (ii) a diagnostic method for detecting the presence of Chlamydia in a biological sample, by contacting the biological sample with an antibody, a polypeptide, or a polypeptide derivative of the invention, such that an immune complex is formed, and by detecting such complex to indicate the presence of Chlamydia in the sample or the organism from which the sample is derived.
  • the immune complex is formed between a component of the sample and the antibody, polypeptide, or polypeptide derivative, whichever is used, and that any unbound material is removed prior to detecting the complex.
  • a polypeptide reagent is useful for detecting the presence of anti- Chlamydia antibodies in a sample, e.g., a blood sample, while an antibody of the invention is used for screening a sample, such as a gastric extract or biopsy, for the presence of Chlamydia polypeptides.
  • the reagent i.e., the antibody, polypeptide, or polypeptide derivative of the invention
  • a solid support such as a tube, a bead, or any other conventional support used in the field. Immobilization is achieved using direct or indirect means. Direct means include passive adsorption (non-covalent binding) or covalent binding between the support and the reagent. By “indirect means” is meant that an anti-reagent compound that interacts with a reagent is first attached to the solid support.
  • an antibody that binds to it can serve as an anti-reagent, provided that it binds to an epitope that is not involved in the recognition of antibodies in biological samples.
  • Indirect means may also employ a ligand-receptor system, for example, where a molecule such as a vitamin is grafted onto the polypeptide reagent and the corresponding receptor immobilized on the solid phase. This is illustrated by the biotin-streptavidin system.
  • a peptide tail is added chemically or by genetic engineering to the reagent and the grafted or fused product immobilized by passive adsorption or covalent linkage of the peptide tail.
  • Such diagnostic agents may be included in a kit which also comprises instructions for use.
  • the reagent is labeled with a detection means which allows for the detection of the reagent when it is bound to its target.
  • the detection means may be a fluorescent agent such as fluorescein isocyanate or fluorescein isothiocyanate, or an enzyme such as horse radish peroxidase or luciferase or alkaline phosphatase, or a radioactive element such as 125 I, or 51 Cr.
  • a tenth aspect of the invention provides a process for purifying, from a biological sample, a polypeptide or polypeptide derivative of the invention, which involves carrying out antibody-based affinity chromatography with the biological sample, wherein the antibody is a monospecific antibody of the invention.
  • the antibody is either polyclonal or monospecific, and preferably is of the IgG type.
  • Purified IgGs is prepared from an antiserum using standard methods (see, e.g., Coligan et al., Current Protocols in Immunology (1994)John Wiley & Sons, Inc., New York, N.Y.). Conventional chromatography supports, as well as standard methods for grafting antibodies, are described in, e.g., Antibodies: A Laboratory Manual, D. Lane, E. Harlow, Eds. (1988) and outlined below.
  • a biological sample such as an C. pneumoniae extract preferably in a buffer solution
  • a chromatography material preferably equilibrated with the buffer used to dilute the biological sample so that the polypeptide or polypeptide derivative of the invention (i.e., the antigen) is allowed to adsorb onto the material.
  • the chromatography material such as a gel or a resin coupled to an antibody of the invention, is in either a batch form or a column.
  • the unbound components are washed off and the antigen is then eluted with an appropriate elution buffer, such as a glycine buffer or a buffer containing a chaotropic agent, e.g., guanidine HCl, or high salt concentration (e.g., 3 M MgCl 2 ).
  • an appropriate elution buffer such as a glycine buffer or a buffer containing a chaotropic agent, e.g., guanidine HCl, or high salt concentration (e.g., 3 M MgCl 2 ).
  • Eluted fractions are recovered and the presence of the antigen is detected, e.g., by measuring the absorbance at 280 nm.
  • An eleventh aspect of the invention provides (i) a composition of matter comprising a monospecific antibody of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a monospecific antibody of the invention, and (iii) a method for treating or preventing a Chlamydia (e.g., C. trachomatis, C. psittaci, C. pneumoniae or C. pecorum ) infection, by administering a therapeutic or prophylactic amount of a monospecific antibody of the invention to an infected individual.
  • a Chlamydia e.g., C. trachomatis, C. psittaci, C. pneumoniae or C. pecorum
  • the eleventh aspect of the invention encompasses the use of a monospecific antibody of the invention in the preparation of a medicament for treating or preventing Chlamydia infection.
  • the monospecific antibody is either polyclonal or monoclonal, preferably of the IgA isotype (predominantly).
  • the antibody is administered to a mucosal surface of a mammal, e.g., the gastric mucosa, e.g., orally or intragastrically, advantageously, in the presence of a bicarbonate buffer.
  • systemic administration not requiring a bicarbonate buffer, is carried out.
  • a monospecific antibody of the invention is administered as a single active component or as a mixture with at least one monospecific antibody specific for a different Chlamydia polypeptide.
  • the amount of antibody and the particular regimen used are readily determined by one skilled in the art. For example, daily administration of about 100 to 1,000 mg of antibodies over one week, or three doses per day of about 100 to 1,000 mg of antibodies over two or three days, are effective regimens for most purposes.
  • Therapeutic or prophylactic efficacy are evaluated using standard methods in the art, e.g., by measuring induction of a mucosal immune response or induction of protective and/or therapeutic immunity, using, e.g., the C. pneumoniae mouse model.
  • the C. pneumoniae strain of the model may be replaced with another Chlamydia strain.
  • the efficacy of DNA molecules and polypeptides from C. pneumoniae is preferably evaluated in a mouse model using C. pneumoniae strain. Protection is determined by comparing the degree of Chlamydia infection to that of a control group. Protection is shown when infection is reduced by comparison to the control group.
  • Such an evaluation is made for polynucleotides, vaccine vectors, polypeptides and derivatives thereof, as well as antibodies of the invention.
  • Adjuvants useful in any of the vaccine compositions described above are as follows.
  • Adjuvants for parenteral administration include aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate. The antigen is precipitated with, or adsorbed onto, the aluminum compound according to standard protocols. Other adjuvants, such as RIBI (ImmunoChem, Hamilton, Mont.), are used in parenteral administration.
  • RIBI ImmunoChem, Hamilton, Mont.
  • Adjuvants for mucosal administration include bacterial toxins, e.g., the cholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridium difficile toxin A and the pertussis toxin (PT), or combinations, subunits, toxoids, or mutants thereof such as a purified preparation of native cholera toxin subunit B (CTB). Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity. Preferably, a mutant having reduced toxicity is used.
  • CT cholera toxin
  • LT E. coli heat-labile toxin
  • PT pertussis toxin
  • CTB native cholera toxin subunit B
  • Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity.
  • a mutant having reduced toxicity is used.
  • Suitable mutants are described, e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/06627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant).
  • Additional LT mutants that are used in the methods and compositions of the invention include, e.g., Ser-63-Lys, Ala-69Gly, Glu-110-Asp, and Glu-112-Asp mutants.
  • Other adjuvants such as a bacterial monophosphoryl lipid A (MPLA) of, e.g., E. coli, Salmonella minnesota, Salmonella typhimurium , or Shigella flexneri ; saponins, or polylactide glycolide (PLGA) microspheres, is also be used in mucosal administration.
  • MPLA bacterial monophosphoryl lipid A
  • Adjuvants useful for both mucosal and parenteral administrations include polyphosphazene (WO 95/02415), DC-chol (3 b-(N-(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol; U.S. Pat. No. 5,283,185 and WO 96/14831) and QS-21 (WO 88/09336).
  • any pharmaceutical composition of the invention containing a polynucleotide, a polypeptide, a polypeptide derivative, or an antibody of the invention is manufactured in a conventional manner.
  • it is formulated with a pharmaceutically acceptable diluent or carrier, e.g., water or a saline solution such as phosphate buffer saline.
  • a diluent or carrier is selected on the basis of the mode and route of administration, and standard pharmaceutical practice.
  • Suitable pharmaceutical carriers or diluents, as well as pharmaceutical necessities for their use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences , a standard reference text in this field and in the USP/NF.
  • the invention also includes methods in which Chlamydia infection are treated by oral administration of a Chlamydia polypeptide of the invention and a mucosal adjuvant, in combination with an antibiotic, an antacid, sucralfate, or a combination thereof.
  • antibiotics including, e.g., macrolides, tetracyclines, and derivatives thereof (specific examples of antibiotics that can be used include azithromycin or doxicyclin or immunomodulators such as cytokines or steroids).
  • compounds containing more than one of the above-listed components coupled together, are used.
  • compositions for carrying out these methods i.e., compositions containing a Chlamydia antigen (or antigens) of the invention, an adjuvant, and one or more of the above-listed compounds, in a pharmaceutically acceptable carrier or diluent.
  • the 60 kDa cysteine rich membrane protein contains a sequence cross-reactive with the murine alpha-myosin heavy chain epitope M7A-alpha, an epitope conserved in humans (Bachmaier et al., Science (1999) 283:1335). This cross-reactivity is proposed to contribute to the development of cardiovascular disease, so it may be beneficial to remove this epitope, and any other epitopes cross-reactive with human antigens, from the protein if it is to be used as a vaccine.
  • a further embodiment of the present invention includes the modification of the coding sequence, for example, by deletion or substitution of the nucleotides encoding the epitope from polynucleotides encoding the protein, as to improve the efficacy and safety of the protein as a vaccine.
  • a similar approach may be appropriate for any protective antigen found to have unwanted homologies or cross-reactivities with human antigens.
  • Amounts of the above-listed compounds used in the methods and compositions of the invention are readily determined by one skilled in the art.
  • Treatment/immunization schedules are also known and readily designed by one skilled in the art.
  • the non-vaccine components can be administered on days 1-14, and the vaccine antigen+adjuvant can be administered on days 7, 14, 21, and 28.
  • the ATP-binding cassette gene was amplified from Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAAT GCGGCCGC CACCATGAAGATGCATAGGCTTAAACC 3′; SEQ ID No:21) and a 3′ primer (5′ GCGCC GGATCC CACTTAAGATATCGATATTTTTGAG 3′; SEQ ID No:22).
  • the 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the ATP-binding cassette protein coding sequence.
  • the 3′ primer includes the sequence encoding the C-terminal sequence of the ATP-binding cassette protein gene and a BamHI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • the PCR fragment was purified using QIAquickTM PCR purification kit (Qiagen), digested with NotI and BamHI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 21) with transcription under control of the human CMV promoter.
  • the secretory locus ORF gene was amplified from Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAAT GCGGCCGC CACCATGCGGTTGGGAAATAAGCCTATGC 3′; SEQ ID No:23) and a 3′ primer (5′ GCGCC GGTACC GTAATTTAATACTCTTTGAAGGGC 3′; SEQ ID No:24).
  • the 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the secretory locus ORF coding sequence.
  • the 3′ primer includes the sequence encoding the C-terminal sequence of the secretory locus ORF protein and a KpnI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • the PCR fragment was purified using QIAquickTM PCR purification kit (Qiagen), digested with NotI and KpnI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 22) with transcription under control of the human CMV promoter.
  • the endopeptidase gene was amplified from Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAAT GCGGCCGC CACCATGCTCACCCTAGGCTTGGAAAGTTCTTG 3′; SEQ ID No:25) and a 3′ primer (5′ GCTTTGGA GGATCC CCGGAGAGGCTAAGGAGAATGG 3′; SEQ ID No:26).
  • the 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the endopeptidase protein coding sequence.
  • the 3′ primer includes the sequence encoding the C-terminal sequence of the endopeptidase protein gene and a BamHI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • the PCR fragment was purified using QIAquickTM PCR purification kit (Qiagen), digested with NotI and BamHI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 23) with transcription under control of the human CMV promoter.
  • the protease gene was amplified from Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAAT GCGGCCGC CACCATGAAAAAAGGGAAATTAGGAGCC 3′; SEQ ID No:27) and a 3′ primer (5′ GCGCC GGATCC CCGAAGCAGAAGTCGTTGTGGG 3′; SEQ ID No:28).
  • the 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the protease protein coding sequence.
  • the 3′ primer includes the sequence encoding the C-terminal sequence of the protease protein gene and a BamHI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • the PCR fragment was purified using QIAquickTM PCR purification kit (Qiagen), digested with NotI and BamHI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 24) with transcription under control of the human CMV promoter.
  • the metalloprotease gene was amplified from Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAAT GCGGCCGC CACCATGAGAAAACTTATTTTATGCAATCCTA 3′; SEQ ID No:29) and a 3′ primer (5′ GCGCC GGATCC CAGAACAACGGAGTTCTTTTGG 3′; SEQ ID No:30).
  • the 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the metalloprotease protein coding sequence.
  • the 3′ primer includes the sequence encoding the C-terminal sequence of the metalloprotease protein gene and a BamHI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • the PCR fragment was purified using QIAquickTM PCR purification kit (Qiagen), digested with NotI and BamHI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 25) with transcription under control of the human CMV promoter.
  • the CLP protease ATPase gene was amplified from Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAAT GCGGCCGC CACCATGAATAAAAAAAATCTAACTATTTG 3′; SEQ ID No:31) and a 3′ primer (5′ GCGCC GGATCC CAGCGATAGCTTCTGGGGTCC 3′; SEQ ID No:32).
  • the 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the CLP protease ATPase protein coding sequence.
  • the 3′ primer includes the sequence encoding the C-terminal sequence of the CLP protease ATPase gene and a BamHI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • the PCR fragment was purified using QIAquickTM PCR purification kit (Qiagen), digested with NotI and BamHI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 26) with transcription under control of the human CMV promoter.
  • the gene encoding CLP protease subunit was amplified from Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAAT GCGGCCGC CACCATGACACTGGTACCCTATGTTG 3′; SEQ ID No:33) and a 3′ primer (5′ GCGCC GGATCC CAGTGCTACTTGTATCCTTATTAG 3′; SEQ ID No:34).
  • the 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the CLP protease subunit coding sequence.
  • the 3′ primer includes the sequence encoding the C-terminal sequence of the CLP protease subunit gene and a BamHI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • the PCR fragment was purified using QIAquickTM PCR purification kit (Qiagen), digested with NotI and BamHI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 27) with transcription under control of the human CMV promoter.
  • the translycolase/transpeptidase gene was amplified from Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAAT GCGGCCGC CACCATGAGCTACCGTAAACGTTCGACTC 3′; SEQ ID No:35) and a 3′ primer (5′ GCGCC GGATCC CCCTCGTTCCCCCTTGTTTCGGAG 3′; SEQ ID No:36).
  • the 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the translycolase/transpeptidase coding sequence.
  • the 3′ primer includes the sequence encoding the C-terminal sequence of the translycolase/transpeptidase gene and a BamHI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • the PCR fragment was purified using QIAquickTM PCR purification kit (Qiagen), digested with NotI and BamHI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 28) with transcription under control of the human CMV promoter.
  • the gene encoding CLPc protease was amplified from Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAAT GCGGCCGC CACCATGTTTGAGAAGTTCACTAATAGAGC 3′; SEQ ID No:37) and a 3′ primer (5′ GCGCC GGTACC GTGATTCCAAGTGAGGGCTAGGG 3′; SEQ ID No:38).
  • the 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the CLPc protease coding sequence.
  • the 3′ primer includes the sequence encoding the C-terminal sequence of the CLPc protease gene and a KpnI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • the PCR fragment was purified using QIAquickTM PCR purification kit (Qiagen), digested with NotI and KpnI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 29) with transcription under control of the human CMV promoter.
  • the thioredoxin gene was amplified from Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAAT GCGGCCGC CACCATGGTAAAGATCATATCAAGTG 3′; SEQ ID No:39) and a 3′ primer (5′ GCGCC GGATCC CAGCGTGCTTATTGATAAG 3′; SEQ ID No:40).
  • the 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the thioredoxin coding sequence.
  • the 3′ primer includes the sequence encoding the C-terminal sequence of the thioredoxin gene and a BamHI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • the PCR fragment was purified using QIAquickTM PCR purification kit (Qiagen), digested with NotI and BamHI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 30) with transcription under control of the human CMV promoter.
  • Plasmid pcDNA3.1( ⁇ )Myc-His C (Invitrogen) was restricted with SpeI and BamHI to remove the CMV promoter and the remaining vector fragment was isolated.
  • the CMV promoter and intron A from plasmid VR-1012 (Vical) was isolated on a SpeI/BamHI fragment. The fragments were ligated together to produce plasmid pCA/Myc-His.
  • DNA concentration was determined by absorbance at 260 nm and the plasmid was verified after gel electrophoresis and ethidium bromide staining by comparison to molecular weight standards. The 5′ and 3′ ends of the gene were verified by sequencing using a LiCor model 4000 L DNA sequencer and IRD-800 labelled primers.
  • mice are susceptible to intranasal infection with different isolates of C. pneumoniae .
  • Strain AR-39 (Grayston et al (1990) Journal of Infectious Diseases 161:618-625) was used in Balb/c mice as a challenge infection model to examine the capacity of Chlamydia gene products delivered as naked DNA to elicit a protective response against a sublethal C. pneumoniae lung infection.
  • Protective immunity is defined as an accelerated clearance of pulmonary infection.
  • mice Groups of 7 to 9 week old male Balb/c mice (8 to 10 per group) were immunized intramuscularly (i.m.) plus intranasally (i.n.) with plasmid DNA containing each of the C. pneumoniae protein gene as described in Examples 1 and 2. Saline or the plasmid vector lacking an inserted Chlamydial gene was given to groups of control animals.
  • Lungs were taken from mice at day 9 post-challenge and immediately homogenised in SPG buffer (7.5% sucrose, 5mM glutamate, 12.5 mM phosphate pH7.5). The homogenate was stored frozen at ⁇ 70° C. until assay. Dilutions of the homogenate were assayed for the presence of infectious Chlamydia by inoculation onto monolayers of susceptible cells. The inoculum was centrifuged onto the cells at 3000 rpm for 1 hour, then the cells were incubated for three days at 35° C. in the presence of 1 ⁇ g/ml cycloheximide.
  • FIG. 31 and Table 1 show that mice immunized i.n. and i.m. with pCACPNM213 had chlamydial lung titers less than 60,000 in 3 of 6 cases at day 9 (mean 51,833) whereas the range of values for control mice sham immunized with saline was 34,200-377,800 IFU/lung (mean 141,450) at day 9.
  • DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM102, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 153,283).
  • the construct pCACPNM102 is identical to pCACPNM213 except that the nucleotide sequence encoding the putative ATP-binding cassette is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated ATP Synthase Subunit I protein.
  • FIG. 32 and Table 3 show that mice immunized i.n. and i.m. with pCACPNM882 had chlamydial lung titers less than 73,000 in 4 of 6 cases at day 9 (mean 77,500) whereas the range of values for control mice sham immunized with saline was 56,000-424,000 IFU/lung (mean 186,291) at day 9.
  • DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM647, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 143,883).
  • the construct pCACPNM647 is identical to pCACPNM882 except that the nucleotide sequence encoding the putative Secretory locus ORF is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated substrate binding protein.
  • FIG. 33 and Table 4 show that mice immunized i.n. and i.m. with pCACPNM208 had chlamydial lung titers less than 67,000 in 4 of 6 cases at day 9 (mean 81,766) whereas the range of values for control mice sham immunized with saline was 56,000-424,100 IFU/lung (mean 186,291) at day 9.
  • DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM647, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 143,883).
  • the construct pCACPNM647 is identical to pCACPNM208 except that the nucleotide sequence encoding the putative Endopeptidase is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated protein.
  • FIG. 34 and Table 6 show that mice immunized i.n. and i.m. with pCACPNM1096 had chlamydial lung titers less than 30,000 in 5 of 6 cases at day 9 (mean 25,000) whereas the range of values for control mice sham immunized with saline was 51,300-170,000 IFU/lung (mean 105,150) at day 9.
  • DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM553, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 111,583).
  • the construct pCACPNM553 is identical to pCACPNM1096 except that the nucleotide sequence encoding the putative Protease is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated protease.
  • the construct pCACPNM1061 is identical to pCACPNM1097 except that the nucleotide sequence encoding the putative Metalloprotease is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated zinc Metalloprotease.
  • FIG. 36 and Table 10 show that mice immunized i.n. and i.m. with pCACPNM908 had chlamydial lung titers less than 40,000 in 3 of 6 cases at day 9 (mean 68,333) whereas the range of values for control mice sham immunized with saline was 56,000-424,100 IFU/lung (mean 207,962) at day 9.
  • DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM569, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 215,600).
  • the construct pCACPNM569 is identical to pCACPNM908 except that the nucleotide sequence encoding the putative CLP protease ATPase is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated signal peptidase.
  • FIG. 37 and Table 12 show that mice immunized i.n. and i.m. with pCACPNM909 had chlamydial lung titers less than 85,000 in 5 of 6 cases at day 9 (mean 87,683) whereas the range of values for control mice sham immunized with saline was 56,000-424,100 IFU/lung (mean 207,962) at day 9.
  • DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM569, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 215,600).
  • pCACPNM569 is identical to pCACPNM909 except that the nucleotide sequence encoding the putative CLP protease subunit is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated signal peptidase.
  • FIG. 38 and Table 14 show that mice immunized i.n. and i.m. with pCACPNM440 had chlamydial lung titers less than 98,000 in 4 of 6 cases at day 9 (mean 87,616) whereas the range of values for control mice sham immunized with saline was 56,000-424,100 IFU/lung (mean 186,291) at day 9. DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM647 failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 143,883).
  • the construct pCACPNM647 is identical to pCACPNM440 except that the nucleotide sequence encoding the putative transglycolase/transpeptidase gene is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated gene.
  • FIG. 39 and Table 16 show that mice immunized i.n. and i.m. with pCACPNM459 had chlamydial lung titers less than 70,000 in 4 of 6 cases at day 9 (mean 70,516) whereas the range of values for control mice sham immunized with saline was 56,000-424,100 IFU/lung (mean 186,291) at day 9.
  • DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM647, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 143,883).
  • the construct pCACPNM647 is identical to pCACPNM459 except that the nucleotide sequence encoding the putative CLPc protease is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated gene.
  • FIG. 40 and Table 18 show that mice immunized i.n. and i.m. with pCACPNM708 had chlamydial lung titers less than 52,000 in 4 of 6 cases at day 9 (mean 73,916) whereas the range of values for control mice sham immunized with saline was 56,000-424,100 IFU/lung (mean 207,962) at day 9.
  • DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM569, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 215,600).
  • pCACPNM569 is identical to pCACPNM708 except that the nucleotide sequence encoding the putative thioredoxin is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated C. pneumoniae gene.
  • This example illustrates the identification of B- and T-cell epitopes in proteins as expressed from each of pCACPNM213, pCACPNM882, pCACPNM208, pCACPNM1096, pCACPNM1097, pCACPNM909, pCACPNM440, pCACPNM459 and pCACPNM708.
  • B-cell epitopes were identified based on the product of flexibilty and hydrophobicity propensities using the program SEQSEE (Wishart D S, et al. “SEQSEE: a comprehensive program suite for protein sequence analysis.” Comput Appl Biosci . 1994 April;10(2):121-32) to identify external surface features (epitopes).
  • T-cell epitopes for HLA-A0201 MHC subclass were identified based on the algorithm of Parker et al. 1995 (Parker K C, et al. “Peptide binding to MHC class I molecules: implications for antigenic peptide prediction.” Immunol Res 1995;14(1):34-57).

Abstract

The present invention provides nucleic acids, proteins and vectors for a method of nucleic acid, including DNA, immunization of a host, including humans, against disease caused by infection by a strain of Chlamydia, specifically C. pneumoniae. The method employs a vector containing a nucleotide sequence encoding a polypeptide of a strain of Chlamydia pneumoniae operably linked to a promoter to effect a expression of the gene product in the host. The polypeptides are derived from C. pneumoniae and are selected from an ATP-binding cassette protein, a secretory locus ORF, an endopeptidase, a protease, a metalloprotease, CLP protease ATPase, a CLP protease subunit, a translycolase/transpeptidase, a CLPc protease and thioredoxin. Modifications are possible within the scope of this invention.

Description

    REFERENCE TO RELATED APPLICATIONS
  • This application claims the benefit of U.S. Provisional Application Nos. 60/202,672, filed May 8, 2000; 60/207,852 filed May 30, 2000; 60/211,801, 60/212,044, 60/211,797, 60/211,796 and 60/211,798 filed Jun. 16, 2000; and 60/235,335, 60/235,361 and 60/235,398 filed Sep. 26, 2000.[0001]
    • FIELD OF INVENTION
  • The present invention relates to a number of [0002] Chlamydia antigens, including an ATP-binding cassette protein, a secretory locus ORF, an endopeptidase, a protease, a metalloprotease, CLP protease ATPase, a CLP protease subunit, a translycolase/transpeptidase, a CLPc protease and thioredoxin, and their corresponding DNA molecules, for the prevention and treatment of Chlamydia infection in mammals.
    • BACKGROUND OF THE INVENTION
  • [0003] Chlamydiae are prokaryotes. They exhibit morphologic and structural similarities to gram-negative bacteria including a trilaminar outer membrane, which contains lipopolysaccharide and several membrane proteins that are structurally and functionally analogous to proteins found in E coli. They are obligate intra-cellular parasites with a unique biphasic life cycle consisting of a metabolically inactive but infectious extracellular stage and a replicating but non-infectious intracellular stage. The replicative stage of the life-cycle takes place within a membrane-bound inclusion which sequesters the bacteria away from the cytoplasm of the infected host cell.
  • [0004] C. pneumoniae is a common human pathogen, originally described as the TWAR strain of Chlamydia psittaci but subsequently recognised to be a new species. C. pneumoniae is antigenically, genetically and morphologically distinct from other Chlamydia species (C. trachomatis, C. pecorum and C. psittaci). It shows 10% or less DNA sequence homology with either of C. trachomatis or C. psittaci.
  • In general, all [0005] chlamydiae share a common developmental microbiology and appear to share a common immunobiology. Genome analysis shown that over 80% of C. pneumoniae and C. trachomatis protein-coding genes are orthologs that share a similar genome organization.
  • [0006] C. pneumoniae is the third most common cause of community acquired pneumonia, only less frequent than Streptococcus pneumoniae and Mycoplasma pneumoniae (Grayston et al. (1995) Journal of Infectious Diseases 168:1231; Campos et al. (1995) Investigation of Ophthalmology and Visual Science 36:1477). It can also cause upper respiratory tract symptoms and disease, including bronchitis and sinusitis (Grayston et al. (1995) Journal of Infectious Diseases 168:1231; Grayston et al (1990) Journal of Infectious Diseases 161:618-625; Marrie (1993) Clinical Infectious Diseases. 18:501-513; Wang et al (1986) Chlamydial infections Cambridge University Press, Cambridge. p. 329. The great majority of the adult population (over 60%) has antibodies to C. pneumoniae (Wang et al (1986) Chlamydial infections. Cambridge University Press, Cambridge. p. 329), indicating past infection which was unrecognized or asymptomatic.
  • [0007] C. pneumoniae infection usually presents as an acute respiratory disease (i.e., cough, sore throat, hoarseness, and fever; abnormal chest sounds on auscultation). For most patients, the cough persists for 2 to 6 weeks, and recovery is slow. In approximately 10% of these cases, upper respiratory tract infection is followed by bronchitis or pneumonia. Furthermore, during a C. pneumoniae epidemic, subsequent co-infection with pneumococcus has been noted in about half of these pneumonia patients, particularly in the infirm and the elderly. As noted above, there is increasing evidence that C. pneumoniae infection is also linked to diseases other than respiratory infections.
  • The reservoir for the organism is presumably people. In contrast to [0008] C. psittaci infections, there is no known bird or animal reservoir. Transmission has not been clearly defined. It may result from direct contact with secretions, from fomites, or from airborne spread. There is a long incubation period, which may last for many months. Based on analysis of epidemics, C. pneumoniae appears to spread slowly through a population (case-to-case interval averaging 30 days) because infected persons are inefficient transmitters of the organism. Susceptibility to C. pneumoniae is universal. Reinfections occur during adulthood, following the primary infection as a child. C. pneumoniae appears to be an endemic disease throughout the world, noteworthy for superimposed intervals of increased incidence (epidemics) that persist for 2 to 3 years. C. trachomatis infection does not confer cross-immunity to C. pneumoniae. Infections are easily treated with oral antibiotics, tetracycline or erythromycin (2 g/d, for at least 10 to 14 d). A recently developed drug, azithromycin, is highly effective as a single-dose therapy against chlamydial infections.
  • In most instances, [0009] C. pneumoniae infection is often mild and without complications, and up to 90% of infections are subacute or unrecognized. Among children in industrialized countries, infections have been thought to be rare up to the age of 5 y, although a recent study (E Normann et al, Chlamydia pneumoniae in children with acute respiratory tract infections, Acta Paediatrica, 1998, Vol 87, Iss 1, pp 23-27) has reported that many children in this age group show PCR evidence of infection despite being seronegative, and estimates a prevalence of 17-19% in 2-4 y olds. In developing countries, the seroprevalence of C. pneumoniae antibodies among young children is elevated, and there are suspicions that C. pneumoniae may be an important cause of acute lower respiratory tract disease and mortality for infants and children in tropical regions of the world.
  • From seroprevalence studies and studies of local epidemics, the initial [0010] C. pneumoniae infection usually happens between the ages of 5 and 20 y. In the USA, for example, there are estimated to be 30,000 cases of childhood pneumonia each year caused by C. pneumoniae. Infections may cluster among groups of children or young adults (e.g., school pupils or military conscripts).
  • [0011] C. pneumoniae causes 10 to 25% of community-acquired lower respiratory tract infections (as reported from Sweden, Italy, Finland, and the USA). During an epidemic, C. pneumonia infection may account for 50 to 60% of the cases of pneumonia. During these periods, also, more episodes of mixed infections with S. pneumoniae have been reported.
  • Reinfection during adulthood is common; the clinical presentation tends to be milder. Based on population seroprevalence studies, there tends to be increased exposure with age, which is particularly evident among men. Some investigators have speculated that a persistent, asymptomatic [0012] C. pneumoniae infection state is common.
  • In adults of middle age or older, [0013] C. pneumoniae infection may progress to chronic bronchitis and sinusitis. A study in the USA revealed that the incidence of pneumonia caused by C. pneumoniae in persons younger than 60 years is 1 case per 1,000 persons per year; but in the elderly, the disease incidence rose three-fold. C. pneumoniae infection rarely leads to hospitalization, except in patients with an underlying illness.
  • Of considerable importance is the association of atherosclerosis and [0014] C. pneumoniae infection. There are several epidemiological studies showing a correlation of previous infections with C. pneumoniae and heart attacks, coronary artery and carotid artery disease (Saikku et al. (1988) Lancet;ii:983-986; Thom et al. (1992) JAMA 268:68-72; Linnanmaki et al. (1993), Circulation 87:1030; Saikku et al. (1992) Annals Internal Medicine 116:273-287; Melnick et al (1993) American Journal of Medicine 95:499). Moreover, the organisms has been detected in atheromas and fatty streaks of the coronary, carotid, peripheral arteries and aorta (Shor et al. (1992) South African. Medical Journal 82:158-161; Kuo et al. (1993) Journal of Infectious Diseases 167:841-849; Kuo et al. (1993) Arteriosclerosis and Thrombosis 13:1501-1504; Campbell et al (1995) Journal of Infectious Diseases 172:585; Chiu et al. Circulation, 1997. Circulation. 96:2144-2148). Viable C. pneumoniae has been recovered from the coronary and carotid artery (Ramirez et al (1996) Annals of Internal Medicine 125:979-982; Jackson et al. 1997. J. Infect. Dis. 176:292-295). Furthermore, it has been shown that C. pneumoniae can induce changes of atherosclerosis in a rabbit model (Fong et al. 1997. Journal of Clinical Microbiolology 35:48 and Laitinen et al. 1997. Infect. Immun. 65:4832-4835). Taken together, these results indicate that it is highly probable that C. pneumoniae can cause atherosclerosis in humans, though the epidemiological importance of chlamydial atherosclerosis remains to be demonstrated.
  • A number of recent studies have also indicated an association between [0015] C. pneumoniae infection and asthma. Infection has been linked to wheezing, asthmatic bronchitis, adult-onset asthma and acute exacerbations of asthma in adults, and small-scale studies have shown that prolonged antibiotic treatment was effective at greatly reducing the severity of the disease in some individuals (Hahn D L, et al. Evidence for Chlamydia pneumoniae infection in steroid-dependent asthma.Ann Allergy Asthma Immunol. 1998 January; 80(1): 45-49.; Hahn D L, et al. Association of Chlamydia pneumoniae IgA antibodies with recently symptomatic asthma. Epidemiol Infect. 1996 December; 117(3): 513-517; Bjornsson E, et al. Serology of chlamydia in relation to asthma and bronchial hyperresponsiveness. Scand J Infect Dis. 1996; 28(1): 63-69.; Hahn D L. Treatment of Chlamydia pneumoniae infection in adult asthma: a before-after trial. J Fam Pract. 1995 October; 41(4): 345-351.; Allegra L, et al. Acute exacerbations of asthma in adults: role of Chlamydia pneumoniae infection. Eur Respir J. 1994 December; 7(12): 2165-2168.; Hahn D L, et al. Association of Chlamydia pneumoniae (strain TWAR) infection with wheezing, asthmatic bronchitis, and adult-onset asthma. JAMA. 1991 July 10; 266(2): 225-230).
  • In light of these results a protective vaccine against [0016] C. pneumoniae infection would be of considerable importance. There is not yet an effective vaccine for any human chlamydial infection. It is conceivable that an effective vaccine can be developed using physically or chemically inactivated Chlamydiae. However, such a vaccine does not have a high margin of safety. In general, safer vaccines are made by genetically manipulating the organism by attenuation or by recombinant means.
  • A disease associated with [0017] C. trachomatis infection is trachoma, a sequela of ocular infection. This disease continues to be a major cause of preventable blindness, with an estimated 500 million cases of active trachoma worldwide (seven million include blindness from conjunctival scarring and eyelid deformities). In the last two decades, genital chlamydial infection has been identified as a major public health problem because of the recognition that chlamydial infection is associated with disease syndromes such as non-gonococcal urethritis, mucopurulent cervicitis, pelvic inflammatory disease (PID), ectopic pregnancy, and tubal infertility. The World Health Organization estimated 89 million new cases of genital chlamydial infections worldwide in 1995. In the United States, each year an estimated four million new cases occur and 50,000 women become infertile as a result of infection.
  • Studies with [0018] C. trachomatis and C. psittaci indicate that safe and effective vaccine against Chlamydia is an attainable goal. For example, mice which have recovered from a lung infection with C. trachomatis are protected from infertility induced by a subsequent vaginal challenge (Pal et al. (1996) Infection and Immunity.64:5341). Similarly, sheep immunized with inactivated C. psittaci were protected from subsequent chlamydial-induced abortions and stillbirths (Jones et al. (1995) Vaccine 13:715). In a mouse model, protection from chlamydial infections has been associated with Th1 immune responses, particularly CD8+ CTL response (Rottenberg et al. 1999. J. Immunol. 162:2829-2836 and Penttila et al. 1999. Immunology. 97:490-496) and it is unlikely that similar responses will need to be induced in humans to confer protection. However, antigens able to elicit a protective immune response against C. pneumoniae are largely unknown. The presence of sufficiently high titres of neutralising antibody at mucosal surfaces can also exert a protective effect (Cotter et al. (1995) Infection and Immunity 63:4704).
  • Antigenic variation within the species [0019] C. pneumoniae is not well documented due to insufficient genetic information, though variation is expected to exist based on C. trachomatis. Serovars of C. trachomatis are defined on the basis of antigenic variation in the major outer membrane protein (MOMP), but published C. pneumoniae MOMP gene sequences show no variation between several diverse isolates of the organism (Campbell et al. Infection and Immunity (1990) 58:93; McCafferty et al Infection and Immunity (1995) 63:2387-9; Gaydos et al. Infection and Immunity.(1992) 60(12):5319-5323). The gene encoding a 76 kDa antigen has been cloned from a single strain of C. pneumoniae and the sequence published (Perez Melgosa et al. Infection and Immunity.(1994) 62:880). An operon encoding the 9 kDa and 60 kDa cyteine-rich outer membrane protein genes has been described (Watson et al., Nucleic Acids Res (1990) 18:5299; Watson et al., Microbiology (1995) 141:2489). Many antigens recognized by immune sera to C. pneumoniae are conserved across all chlamydiae, but 98 kDa, 76 kDa and several other proteins may be C. pneumoniae-specific (Knudsen et al. Infect. Immun. 1999. 67:375-383; Perez Melgosa et al. Infection and Immunity. 1994. 62:880; Melgosa et al., FEMS Microbiol Lett 1993. 112:199;, Campbell et al., J. Clin. Microbiol. 1990. 28 :1261; Iijima et al., J. Clin. Microbiol. 1994. 32:583). Antisera to 76 kDa and 54 kDa antigens have been reported to neutralize C. pneumoniae in vitro (Perez Melgosa et al. 1994. Infect. Immun. 62:880-886 and Wiedman-Al-Ahmad et al. 1997. Clin. Diagn. Lab. Immunol. 4:700-704). An assessment of the number and relative frequency of any C. pneumoniae serotypes, and the defining antigens, is not yet possible. The entire genome sequence of C. pneumoniae strain CWL-029 is now known (http://chlamydia-www.berkeley.edu:4231/) and as further sequences become available a better understanding of antigenic variation may be gained.
  • Many antigens recognised by immune sera to [0020] C. pneumoniae are conserved across all chlamydiae, but 98kDa, 76 kDa and 54 kDa proteins appear to be C. pneumoniae-specific (Campos et al. (1995) Investigation of Ophthalmology and Visual Science 36:1477; Marrie (1993) Clinical Infectious Diseases. 18:501-513; Wiedmann-Al-Ahmad M, et al. Reactions of polyclonal and neutralizing anti-p54 monoclonal antibodies with an isolated, species-specific 54-kilodalton protein of Chlamydia pneumoniae. Clin Diagn Lab Immunol. 1997 Nov.; 4(6): 700-704).
  • Immunoblotting of isolates with sera from patients does show variation of blotting patterns between isolates, indicating that serotypes [0021] C. pneumoniae may exist (Grayston et al. (1995) Journal of Infectious Diseases 168:1231; Ramirez et al (1996) Annals of Internal Medicine 125:979-982). However, the results are potentially confounded by the infection status of the patients, since immunoblot profiles of a patient's sera change with time post-infection. An assessment of the number and relative frequency of any serotypes, and the defining antigens, is not yet possible.
  • The use of DNA immunization to elicit a protective immune response in Balb/c mice against pulmonary infection with the mouse pneumonitis (MoPn) strain of [0022] Chlamydia trachomatis has recently been described (Zhang et al. 1997. J. Infect. Dis. 76:1035-1040 and Zhang et al. 1999. Immunology. 96:314-321). Recently the genome sequence from C. pneumoniae strain CM1 (ATCC #1360-VR) has been disclosed by Griffais in WO99/27105 on Jun. 3, 1999.
  • Accordingly, a need exists for identifying and isolating polynucleotide sequences of [0023] C. pneumoniae for use in preventing and treating Chlamydia infection.
    • SUMMARY OF THE INVENTION
  • The present invention provides purified and isolated polynucleotide molecules that encode a [0024] Chlamydia polypeptide selected from: an ATP-binding cassette protein, a secretory locus ORF, an endopeptidase, a protease, a metalloprotease, CLP protease ATPase, a CLP protease subunit, a translycolase/transpeptidase, a CLPc protease and thioredoxin. The polynucleotide molecules can be used in methods to prevent, treat, and diagnose Chlamydia infection. In one embodiment of the invention, the polynucleotide molecules is DNA that encode a polypeptide of any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
  • Another form of the invention provides polypeptides corresponding to an isolated DNA molecule. Amino acid sequences of the corresponding encoded polypeptides are shown in one embodiment as SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20. [0025]
  • Those skilled in the art will readily understand that the invention, having provided the polynucleotide sequences encoding [0026] Chlamydia polypeptides, also provides polynucleotides encoding fragments derived from such polypeptides. Moreover, the invention is understood to provide mutants and derivatives of such polypeptides and fragments derived therefrom, which result from the addition, deletion, or substitution of non-essential amino acids as described herein. Those skilled in the art would also readily understand that the invention, having provided the polynucleotide sequences encoding Chlamydia polypeptides, further provides monospecific antibodies that specifically bind to such polypeptides.
  • The present invention has wide application and includes expression cassettes, vectors, and cells transformed or transfected with the polynucleotides of the invention. Accordingly, the present invention further provides (i) a method for producing a polypeptide of the invention in a recombinant host system and related expression cassettes, vectors, and transformed or transfected cells; (ii) a vaccine, or a live vaccine vector such as a pox virus, [0027] Salmonella typhimurium, or Vibrio cholerae vector, containing a polypeptide or a polynucleotide of the invention, such vaccines and vaccine vectors being useful for, e.g., preventing and treating Chlamydia infection, in combination with a diluent or carrier, and related pharmaceutical compositions and associated therapeutic and/or prophylactic methods; (iii) a therapeutic and/or prophylactic use of an RNA or DNA molecule of the invention, either in a naked form or formulated with a delivery vehicle, a polypeptide or combination of polypeptides, or a monospecific antibody of the invention, and related pharmaceutical compositions; (iv) a method for diagnosing the presence of Chlamydia in a biological sample, which can involve the use of a DNA or RNA molecule, a monospecific antibody, or a polypeptide of the invention; and (v) a method for purifying a polypeptide of the invention by antibody-based affinity chromatography.
  • One aspect of the invention provides a vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of: [0028]
  • (i) a nucleic acid sequence set forth in any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19; [0029]
  • (ii) a nucleic acid sequence which encodes a polypeptide encoded by any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19; [0030]
  • (iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19; and [0031]
  • (iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20; [0032]
  • (v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv); [0033]
  • wherein each first nucleic acid is capable of being expressed. [0034]
  • Another aspect of the invention provides a vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of: [0035]
  • (i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19; [0036]
  • (ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20; [0037]
  • (iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii); [0038]
  • wherein each first nucleic acid is capable of being expressed.[0039]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • The present invention will be further understood from the following description with reference to the drawings, in which: [0040]
  • FIG. 1 shows the nucleotide sequence of the gene encoding an ATP-binding cassette (SEQ ID No: 1) and the deduced amino acid sequence of the ATP-binding cassette from [0041] Chlamydia pneumoniae (SEQ ID No: 2).
  • FIG. 2 shows the nucleotide sequence of the gene encoding a secretory locus ORF (SEQ ID No: 3) and the deduced amino acid sequence of the secretory locus ORF from [0042] Chlamydia pneumoniae (SEQ ID No: 4).
  • FIG. 3 shows the nucleotide sequence of the gene encoding an endopeptidase (SEQ ID No: 5) and the deduced amino acid sequence of the endopeptidase from [0043] Chlamydia pneumoniae (SEQ ID No: 6).
  • FIG. 4 shows the nucleotide sequence of the gene encoding a protease (SEQ ID No: 7) and the deduced amino acid sequence of the protease from [0044] Chlamydia pneumoniae (SEQ ID No: 8).
  • FIG. 5 shows the nucleotide sequence of the gene encoding a metalloprotease (SEQ ID No: 9) and the deduced amino acid sequence of the metalloprotease from [0045] Chlamydia pneumoniae (SEQ ID No: 10).
  • FIG. 6 shows the nucleotide sequence of the gene encoding CLP protease ATPase (SEQ ID No: 11) and the deduced amino acid sequence of the CLP protease ATPase from [0046] Chlamydia pneumoniae (SEQ ID No: 12).
  • FIG. 7 shows the nucleotide sequence of the gene encoding a CLP protease subunit (SEQ ID No: 13) and the deduced amino acid sequence of the CLP protease subunit from [0047] Chlamydia pneumoniae (SEQ ID No: 14).
  • FIG. 8 shows the nucleotide sequence of the gene encoding a translycolase/transpeptidase (SEQ ID No: 15) and the deduced amino acid sequence of the transglycolase/transpeptidase from [0048] Chlamydia pneumoniae (SEQ ID No: 16).
  • FIG. 9 shows the nucleotide sequence of the gene encoding a CLPc protease (SEQ ID No: 17) and the deduced amino acid sequence of the CLPc protease from [0049] Chlamydia pneumoniae (SEQ ID No: 18).
  • FIG. 10 shows the nucleotide sequence of the gene encoding thioredoxin (SEQ ID No: 19) and the deduced amino acid sequence of thioredoxin from [0050] Chlamydia pneumoniae (SEQ ID No: 20).
  • FIG. 11 shows the restriction enzyme analysis of the [0051] C. pneumoniae gene encoding an ATP-binding cassette.
  • FIG. 12 shows shows the restriction enzyme analysis of the [0052] C. pneumoniae gene encoding a secretory locus ORF.
  • FIG. 13 shows the restriction enzyme analysis of the [0053] C. pneumoniae gene encoding an endopeptidase.
  • FIG. 14 shows the restriction enzyme analysis of the [0054] C. pneumoniae gene encoding a protease.
  • FIG. 15 shows the restriction enzyme analysis of the [0055] C. pneumoniae gene encoding a metalloprotease.
  • FIG. 16 shows the restriction enzyme analysis of the [0056] C. pneumoniae gene encoding CLP protease ATPase.
  • FIG. 17 shows the restriction enzyme analysis of the [0057] C. pneumoniae gene encoding a CLP protease subunit.
  • FIG. 18 shows the restriction enzyme analysis of the [0058] C. pneumoniae gene encoding a translycolase/transpeptidase.
  • FIG. 19 shows the restriction enzyme analysis of the [0059] C. pneumoniae gene encoding a CLPc protease.
  • FIG. 20 shows the restriction enzyme analysis of the [0060] C. pneumoniae gene encoding thioredoxin.
  • FIG. 21 shows the construction and elements of plasmid pCACPNM213. [0061]
  • FIG. 22 shows the construction and elements of plasmid pCACPNM882. [0062]
  • FIG. 23 shows the construction and elements of plasmid pCACPNM208. [0063]
  • FIG. 24 shows the construction and elements of plasmid pCACPNM1096. [0064]
  • FIG. 25 shows the construction and elements of plasmid pCACPNM1097. [0065]
  • FIG. 26 shows the construction and elements of plasmid pCACPNM908. [0066]
  • FIG. 27 shows the construction and elements of plasmid pCACPNM909. [0067]
  • FIG. 28 shows the construction and elements of plasmid pCACPNM440. [0068]
  • FIG. 29 shows the construction and elements of plasmid pCACPNM459. [0069]
  • FIG. 30 shows the construction and elements of plasmid pCACPNM708. [0070]
  • FIG. 31 illustrates protection against [0071] C. pneumoniae infection by pCACPNM213 following DNA immunization.
  • FIG. 32 illustrates protection against [0072] C. pneumoniae infection by pCACPNM882 following DNA immunization.
  • FIG. 33 illustrates protection against [0073] C. pneumoniae infection by pCACPNM208 following DNA immunization.
  • FIG. 34 illustrates protection against [0074] C. pneumoniae infection by pCACPNM1096 following DNA immunization.
  • FIG. 35 illustrates protection against [0075] C. pneumoniae infection by pCACPNM1097 following DNA immunization.
  • FIG. 36 illustrates protection against [0076] C. pneumoniae infection by pCACPNM908 following DNA immunization.
  • FIG. 37 illustrates protection against [0077] C. pneumoniae infection by pCACPNM909 following DNA immunization.
  • FIG. 38 illustrates protection against [0078] C. pneumoniae infection by pCACPNM2440 following DNA immunization.
  • FIG. 39 illustrates protection against [0079] C. pneumoniae infection by pCACPNM459 following DNA immunization.
  • FIG. 40 illustrates protection against [0080] C. pneumoniae infection by pCACPNM708 following DNA immunization.
    • DETAILED DESCRIPTION OF INVENTION
  • Open reading frames (ORFs) encoding a number of Chlamydial proteins have been identified from the [0081] C. pneumoniae genome. These proteins include an ATP-binding cassette protein, a secretory locus ORF, an endopeptidase, a protease, a metalloprotease, CLP protease ATPase, a CLP protease subunit, a translycolase/transpeptidase, a CLPc protease and thioredoxin. The gene encoding each of these polypeptides has been inserted into an expression plasmid and shown to confer immune protection against chlamydial infection. Accordingly, any one of these and related polypeptides can be used to prevent and treat Chlamydia infection.
  • According to a first aspect of the invention, isolated polynucleotides are provided which encode [0082] Chlamydia polypeptides, whose amino acid sequences are shown in SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
  • The term “isolated polynucleotide” is defined as a polynucleotide removed from the environment in which it naturally occurs. For example, a naturally-occurring DNA molecule present in the genome of a living bacteria or as part of a gene bank is not isolated, but the same molecule separated from the remaining part of the bacterial genome, as a result of, e.g., a cloning event (amplification), is isolated. Typically, an isolated DNA molecule is free from DNA regions (e.g., coding regions) with which it is immediately contiguous at the 5′ or 3′ end, in the naturally occurring genome. Such isolated polynucleotides may be part of a vector or a composition and still be defined as isolated in that such a vector or composition is not part of the natural environment of such polynucleotide. [0083]
  • The polynucleotide of the invention is either RNA or DNA (cDNA, genomic DNA, or synthetic DNA), or modifications, variants, homologs or fragments thereof. The DNA is either double-stranded or single-stranded, and, if single-stranded, is either the coding strand or the non-coding (anti-sense) strand. Any one of the sequences that encode the polypeptides of the invention as shown in any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 is (a) a coding sequence, (b) a ribonucleotide sequence derived from transcription of (a), or (c) a coding sequence which uses the redundancy or degeneracy of the genetic code to encode the same polypeptides. By “polypeptide” or “protein” is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Both terms are used interchangeably in the present application. [0084]
  • Consistent with the first aspect of the invention, amino acid sequences are provided which are homologous to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20. As used herein, “homologous amino acid sequence” is any polypeptide which is encoded, in whole or in part, by a nucleic acid sequence which hybridizes at 25-35° C. below critical melting temperature (Tm), to any portion of the nucleic acid sequence of any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. A homologous amino acid sequence is one that differs from an amino acid sequence shown in any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 by one or more conservative amino acid substitutions. Such a sequence also encompass serotypic variants (defined below) as well as sequences containing deletions or insertions which retain inherent characteristics of the polypeptide such as immunogenicity. Preferably, such a sequence is at least 75%, preferably at least 78%, more preferably at least 80%, even more preferably at least 85%, 88% or 90%, and most preferably at least 93%, 95% or 98% identical to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20. [0085]
  • Homologous amino acid sequences include sequences that are identical or substantially identical to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20. By “amino acid sequence substantially identical” is meant a sequence that is at least 90%, preferably 95%, more preferably 97%, and most preferably 99% identical to an amino acid sequence of reference and that preferably differs from the sequence of reference by a majority of conservative amino acid substitutions. [0086]
  • Conservative amino acid substitutions are substitutions among amino acids of the same class. These classes include, for example, amino acids having uncharged polar side chains, such as asparagine, glutamine, serine, threonine, and tyrosine; amino acids having basic side chains, such as lysine, arginine, and histidine; amino acids having acidic side chains, such as aspartic acid and glutamic acid; and amino acids having nonpolar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine. [0087]
  • Homology is measured using sequence analysis software such as Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705. Amino acid sequences are aligned to maximize identity. Gaps may be artificially introduced into the sequence to attain proper alignment. Once the optimal alignment has been set up, the degree of homology is established by recording all of the positions in which the amino acids of both sequences are identical, relative to the total number of positions. [0088]
  • Homologous polynucleotide sequences are defined in a similar way. Preferably, a homologous sequence is one that is at least 45%, more preferably 50%, 55%, 60%, 65%, 70%, 75%, 80%, and even more preferably 85%, 87%, 90%, 93%, 96% and most preferably 99% identical to the coding sequence of any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. [0089]
  • Consistent with the first aspect of the invention, polypeptides having a sequence homologous to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 include naturally-occurring allelic variants, as well as mutants or any other non-naturally occurring variants that retain the inherent characteristics of the polypeptide of any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20. [0090]
  • As is known in the art, an allelic variant is an alternate form of a polypeptide that is characterized as having a substitution, deletion, or addition of one or more amino acids that does not alter the biological function of the polypeptide. By “biological function” is meant the function of the polypeptide in the cells in which it naturally occurs, even if the function is not necessary for the growth or survival of the cells. For example, the biological function of a porin is to allow the entry into cells of compounds present in the extracellular medium. Biological function is distinct from antigenic property. A polypeptide can have more than one biological function. [0091]
  • Allelic variants are very common in nature. For example, a bacterial species such as [0092] C. pneumoniae, is usually represented by a variety of strains that differ from each other by minor allelic variations. Indeed, a polypeptide that fulfills the same biological function in different strains can have an amino acid sequence (and polynucleotide sequence) that is not identical in each of the strains. Despite this variation, an immune response directed generally against many allelic variants has been demonstrated. In studies of the Chlamydial MOMP antigen, cross-strain antibody binding plus neutralization of infectivity occurs despite amino acid sequence variation of MOMP from strain to strain, indicating that the MOMP, when used as an immunogen, is tolerant of amino acid variations.
  • Polynucleotides encoding homologous polypeptides or allelic variants are retrieved by polymerase chain reaction (PCR) amplification of genomic bacterial DNA extracted by conventional methods. This involves the use of synthetic oligonucleotide primers matching upstream and downstream of the 5′ and 3′ ends of the encoding domain. Suitable primers are designed according to the nucleotide sequence information provided in any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. The procedure is as follows: a primer is selected which consists of 10 to 40, preferably 15 to 25 nucleotides. It is advantageous to select primers containing C and G nucleotides in a proportion sufficient to ensure efficient hybridization; i.e., an amount of C and G nucleotides of at least 40%, preferably 50% of the total nucleotide content. A standard PCR reaction contains typically 0.5 to 5 Units of Taq DNA polymerase per 100 μL, 20 to 200 μM deoxynucleotide each, preferably at equivalent concentrations, 0.5 to 2.5 mM magnesium over the total deoxynucleotide concentration, 10[0093] 5 to 106 target molecules, and about 20 pmol of each primer. About 25 to 50 PCR cycles are performed, with an annealing temperature 15° C. to 5° C. below the true Tm of the primers. A more stringent annealing temperature improves discrimination against incorrectly annealed primers and reduces incorportion of incorrect nucleotides at the 3′ end of primers. A denaturation temperature of 95° C. to 97° C. is typical, although higher temperatures may be appropriate for dematuration of G+C−rich targets. The number of cycles performed depends on the starting concentration of target molecules, though typically more than 40 cycles is not recommended as non-specific background products tend to accumulate.
  • An alternative method for retrieving polynucleotides encoding homologous polypeptides or allelic variants is by hybridization screening of a DNA or RNA library. Hybridization procedures are well-known in the art and are described in Ausubel et al., (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994), Silhavy et al. (Silhavy et al. Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, 1984), and Davis et al. (Davis et al. A Manual for Genetic Engineering: Advanced Bacterial Genetics, Cold Spring Harbor Laboratory Press, 1980)). Important parameters for optimizing hybridization conditions are reflected in a formula used to obtain the critical melting temperature above which two complementary DNA strands separate from each other (Casey & Davidson, Nucl. Acid Res. (1977) 4:1539). For polynucleotides of about 600 nucleotides or larger, this formula is as follows: Tm=81.5+0.41×(% G+C)+16.6 log (cation ion concentration)−0.63×(% formamide)−600/base number. Under appropriate stringency conditions, hybridization temperature (Th) is approximately 20 to 40° C., 20 to 25° C., or, preferably 30 to 40° C. below the calculated Tm. Those skilled in the art will understand that optimal temperature and salt conditions can be readily determined. [0094]
  • For the polynucleotides of the invention, stringent conditions are achieved for both pre-hybridizing and hybridizing incubations (i) within 4-16 hours at 42° C., in 6×SSC containing 50% formamide, or (ii) within 4-16 hours at 65° C. in an aqueous 6×SSC solution (1 M NaCl, 0.1 M sodium citrate (pH 7.0)). Typically, hybridization experiments are performed at a temperature from 60 to 68° C., e.g. 65° C. At such a temperature, stringent hybridization conditions can be achieved in 6×SSC, preferably in 2×SSC or 1×SSC, more preferably in 0.5×SSc, 0.3×SSC or 0.1×SSC (in the absence of formamide). 1×SSC contains 0.15 M NaCl and 0.015 M sodium citrate. [0095]
  • Useful homologs and fragments thereof that do not occur naturally are designed using known methods for identifying regions of an antigen that are likely to tolerate amino acid sequence changes and/or deletions. As an example, homologous polypeptides from different species are compared; conserved sequences are identified. The more divergent sequences are the most likely to tolerate sequence changes. Homology among sequences may be analyzed using, as an example, the BLAST homology searching algorithm of Altschul et al., Nucleic Acids Res.; 25:3389-3402 (1997). Alternatively, sequences are modified such that they become more reactive to T- and/or B-cells, based on computer-assisted analysis of probable T- or B-cell epitopes Yet another alternative is to mutate a particular amino acid residue or sequence within the polypeptide in vitro, then screen the mutant polypeptides for their ability to prevent or treat [0096] Chlamydia infection according to the method outlined below.
  • A person skilled in the art will readily understand that by following the screening process of this invention, it will be determined without undue experimentation whether a particular homolog of any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 may be useful in the prevention or treatment of [0097] Chlamydia infection. The screening procedure comprises the steps:
  • (i) immunizing an animal, preferably mouse, with the test homolog or fragment; [0098]
  • (ii) inoculating the immunized animal with [0099] Chlamydia; and
  • (iii) selecting those homologs or fragments which confer protection against [0100] Chlamydia.
  • By “conferring protection” is meant that there is a reduction in severity of any of the effects of [0101] Chlamydia infection, in comparison with a control animal which was not immunized with the test homolog or fragment.
  • Consistent with the first aspect of the invention, polypeptide derivatives are provided that are partial sequences of any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, partial sequences of polypeptide sequences homologous to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, polypeptides derived from full-length polypeptides by internal deletion, and fusion proteins. [0102]
  • It is an accepted practice in the field of immunology to use fragments and variants of protein immunogens as vaccines, as all that is required to induce an immune response to a protein is a small (e.g., 8 to 10 amino acid) immunogenic region of the protein. Various short synthetic peptides corresponding to surface-exposed antigens of pathogens other than [0103] Chlamydia have been shown to be effective vaccine antigens against their respective pathogens, e.g. an 11 residue peptide of murine mammary tumor virus (Casey & Davidson, Nucl. Acid Res. (1977) 4:1539), a 16-residue peptide of Semliki Forest virus (Snijders et al., 1991. J. Gen. Virol. 72:557-565), and two overlapping peptides of 15 residues each from canine parvovirus (Langeveld et al., Vaccine 12(15):1473-1480, 1994)
  • Accordingly, it will be readily apparent to one skilled in the art, having read the present description, that partial sequences of any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 or their homologous amino acid sequences are inherent to the full-length sequences and are taught by the present invention. Such polypeptide fragments preferably are at least 12 amino acids in length. Advantageously, they are at least 15 amino acids, preferably at least 20, 25, 30, 35, 40, 45, 50 amino acids, more preferably at least 55, 60, 65, 70, 75 amino acids, and most preferably at least 80, 85, 90, 95, 100 amino acids in length. [0104]
  • Polynucleotides of 30 to 600 nucleotides encoding partial sequences of sequences homologous to any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 are retrieved by PCR amplification using the parameters outlined above and using primers matching the sequences upstream and downstream of the 5′ and 3′ ends of the fragment to be amplified. The template polynucleotide for such amplification is either the full length polynucleotide homologous to any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, or a polynucleotide contained in a mixture of polynucleotides such as a DNA or RNA library. As an alternative method for retrieving the partial sequences, screening hybridization is carried out under conditions described above and using the formula for calculating Tm. Where fragments of 30 to 600 nucleotides are to be retrieved, the calculated Tm is corrected by subtracting (600/polynucleotide size in base pairs) and the stringency conditions are defined by a hybridization temperature that is 5 to 10° C. below Tm. Where oligonucleotides shorter than 20-30 bases are to be obtained, the formula for calculating the Tm is as follows: Tm=4×(G+C)+2 (A+T). For example, an 18 nucleotide fragment of 50% G+C would have an approximate Tm of 54° C. Short peptides that are fragments of any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 or its homologous sequences, are obtained directly by chemical synthesis (E. Gross and H. J. Meinhofer, 4 The Peptides: Analysis, Synthesis, Biology; Modern Techniques of Peptide Synthesis, John Wiley & Sons (1981), and M. Bodanzki, Principles of Peptide Synthesis, Springer-Verlag (1984)). [0105]
  • Useful polypeptide derivatives, e.g., polypeptide fragments, are designed using computer-assisted analysis of amino acid sequences. This would identify probable surface-exposed, antigenic regions (Hughes et al., 1992. Infect. Immun. 60(9):3497). Analysis of 6 amino acid sequences contained in any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, based on the product of flexibility and hydrophobicity propensities using the program SEQSEE (Wishart D S, et al. “SEQSEE: a comprehensive program suite for protein sequence analysis.” [0106] Comput Appl Biosci. 1994 April;10(2):121-32), reveal potential B- and T-cell epitopes which may be used as a basis for selecting useful immunogenic fragments and variants. This analysis uses a reasonable combination of external surface features that is likely to be recognized by antibodies. Probable T-cell epitopes for HLA-A0201 MHC subclass may be revealed by an algorithms that emulate an approach developed at the NIH (Parker K C, et al. “Peptide binding to MHC class I molecules: implications for antigenic peptide prediction.” Immunol Res 1995;14(1):34-57). The potential B-cell and T-cell epitopes are shown in Tables 2, 5, 7, 9, 11, 13, 15, 17 and 19 and SEQ ID NOs: 41 to 74. Sequences which are substantially identical to SEQ ID NOS: 41 to 74, or which are conservatively substituted variants of SEQ ID NOs: 41 to 74, are expected to be functional epitopes and are within the scope of the invention.
  • Epitopes which induce a protective T cell-dependent immune response are present throughout the length of the polypeptide. However, some epitopes may be masked by secondary and tertiary structures of the polypeptide. To reveal such masked epitopes large internal deletions are created which remove much of the original protein structure and exposes the masked epitopes. Such internal deletions sometimes effect the additional advantage of removing immunodominant regions of high variability among strains. [0107]
  • Polynucleotides encoding polypeptide fragments and polypeptides having large internal deletions are constructed using standard methods (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994). Such methods include standard PCR, inverse PCR, restriction enzyme treatment of cloned DNA molecules, or the method of Kunkel et al. (Kunkel et al. Proc. Natl. Acad. Sci. USA (1985) 82:448). Components for these methods and instructions for their use are readily available from various commercial sources such as Stratagene. Once the deletion mutants have been constructed, they are tested for their ability to prevent or treat [0108] Chlamydia infection as described above.
  • As used herein, a fusion polypeptide is one that contains a polypeptide or a polypeptide derivative of the invention fused at the N- or C-terminal end to any other polypeptide (hereinafter referred to as a peptide tail). A simple way to obtain such a fusion polypeptide is by translation of an in-frame fusion of the polynucleotide sequences, i.e., a hybrid gene. The hybrid gene encoding the fusion polypeptide is inserted into an expression vector which is used to transform or transfect a host cell. Alternatively, the polynucleotide sequence encoding the polypeptide or polypeptide derivative is inserted into an expression vector in which the polynucleotide encoding the peptide tail is already present. Such vectors and instructions for their use are commercially available, e.g. the pMal-c2 or pMal-p2 system from New England Biolabs, in which the peptide tail is a maltose binding protein, the glutathione-S-transferase system of Pharmacia, or the His-Tag system available from Novagen. These and other expression systems provide convenient means for further purification of polypeptides and derivatives of the invention. [0109]
  • An advantageous example of a fusion polypeptide is one where the polypeptide or homolog or fragment of the invention is fused to a polypeptide having adjuvant activity, such as subunit B of either cholera toxin or [0110] E. coli heat-labile toxin. Another advantageous fusion is one where the polypeptide, homolog or fragment is fused to a strong T-cell epitope or B-cell epitope. Such an epitope may be one known in the art (e.g. the Hepatitis B virus core antigen, D. R. Millich et al., “Antibody production to the nucleocapsid and envelope of the Hepatitis B virus primed by a single synthetic T cell site”, Nature. 1987. 329:547-549), or one which has been identified in another polypeptide of the invention based on computer-assisted analysis of probable T- or B-cell epitopes. Consistent with this aspect of the invention is a fusion polypeptide comprising T- or B-cell epitopes from any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 or its homolog or fragment, wherein the epitopes are derived from multiple variants of said polypeptide or homolog or fragment, each variant differing from another in the location and sequence of its epitope within the polypeptide. Such a fusion is effective in the prevention and treatment of Chlamydia infection since it optimizes the T- and B-cell response to the overall polypeptide, homolog or fragment.
  • To effect fusion, the polypeptide of the invention is fused to the N-, or preferably, to the C-terminal end of the polypeptide having adjuvant activity or T- or B-cell epitope. Alternatively, a polypeptide fragment of the invention is inserted internally within the amino acid sequence of the polypeptide having adjuvant activity. The T- or B-cell epitope may also be inserted internally within the amino acid sequence of the polypeptide of the invention. [0111]
  • Consistent with the first aspect, the polynucleotides of the invention also encode hybrid-precursor polypeptides containing heterologous signal peptides, which mature into polypeptides of the invention. By “heterologous signal peptide” is meant a signal peptide that is not found in naturally-occurring precursors of polypeptides of the invention. [0112]
  • Polynucleotide molecules according to the invention, including RNA, DNA, or modifications or combinations thereof, have various applications. A DNA molecule is used, for example, (i) in a process for producing the encoded polypeptide in a recombinant host system, (ii) in the construction of vaccine vectors such as poxviruses, which are further used in methods and compositions for preventing and/or treating [0113] Chlamydia infection, (iii) as a vaccine agent (as well as an RNA molecule), in a naked form or formulated with a delivery vehicle and, (iv) in the construction of attenuated Chlamydia strains that can over-express a polynucleotide of the invention or express it in a non-toxic, mutated form.
  • Selected genes from pathogenic micro-organisms within an eukaryotic expression plasmid are useful as vaccines. Expression plasmids contain methylated CpG motifs that elicit innate cytokine responses that promote the canalization of CD4 T cell responses to a Thl cytokine secretion pattern. The intracellular synthesis of the microbial protein, especially within transfected professional antigen-presenting cells, facilitates the presentation of antigen on class I and class II molecules and the induction of cell-mediated immunity. The use of one or a number of microbial protein-coding genes allows the presentation of protective antigens to the immune system to occur in the absence of microbe-directed immune evasion mechanisms and in the absence of competing or pathologic antigens. Immune responses primed by DNA vaccines are also readily amplified by protein-antigen immunization. Thus, immunization with DNA vaccines is particularly relevant to chlamydial vaccine design. [0114]
  • Accordingly, a second aspect of the invention encompasses (i) an expression cassette containing a DNA molecule of the invention placed under the control of the elements required for expression, in particular under the control of an appropriate promoter; (ii) an expression vector containing an expression cassette of the invention; (iii) a procaryotic or eucaryotic cell transformed or transfected with an expression cassette and/or vector of the invention, as well as (iv) a process for producing a polypeptide or polypeptide derivative encoded by a polynucleotide of the invention, which involves culturing a procaryotic or eucaryotic cell transformed or transfected with an expression cassette and/or vector of the invention, under conditions that allow expression of the DNA molecule of the invention and, recovering the encoded polypeptide or polypeptide derivative from the cell culture. [0115]
  • A recombinant expression system is selected from procaryotic and eucaryotic hosts. Eucaryotic hosts include yeast cells (e.g., [0116] Saccharomyces cerevisiae or Pichia pastoris), mammalian cells (e.g., COS1, NIH3T3, or JEG3 cells), arthropods cells (e.g., Spodoptera frugiperda (SF9) cells), and plant cells. A preferred expression system is a procaryotic host such as E. coli. Bacterial and eucaryotic cells are available from a number of different sources including commercial sources to those skilled in the art, e.g., the American Type Culture Collection (ATCC; Rockville, Md.). Commercial sources of cells used for recombinant protein expression also provide instructions for usage of the cells.
  • The choice of the expression system depends on the features desired for the expressed polypeptide. For example, it may be useful to produce a polypeptide of the invention in a particular lipidated form or any other form. [0117]
  • One skilled in the art would redily understand that not all vectors and expression control sequences and hosts would be expected to express equally well the polynucleotides of this invention. With the guidelines described below, however, a selection of vectors, expression control sequences and hosts may be made without undue experimentation and without departing from the scope of this invention. [0118]
  • In selecting a vector, the host must be chosen that is compatible with the vector which is to exist and possibly replicate in it. Considerations are made with respect to the vector copy number, the ability to control the copy number, expression of other proteins such as antibiotic resistance. In selecting an expression control sequence, a number of variables are considered. Among the important variable are the relative strength of the sequence (e.g. the ability to drive expression under various conditions), the ability to control the sequence's function, compatibility between the polynucleotide to be expressed and the control sequence (e.g. secondary structures are considered to avoid hairpin structures which prevent efficient transcription). In selecting the host, unicellular hosts are selected which are compatible with the selected vector, tolerant of any possible toxic effects of the expressed product, able to secrete the expressed product efficiently if such is desired, to be able to express the product in the desired conformation, to be easily scaled up, and to which ease of purification of the final product. [0119]
  • The choice of the expression cassette depends on the host system selected as well as the features desired for the expressed polypeptide. Typically, an expression cassette includes a promoter that is functional in the selected host system and can be constitutive or inducible; a ribosome binding site; a start codon (ATG) if necessary; a region encoding a signal peptide, e.g., a lipidation signal peptide; a DNA molecule of the invention; a stop codon; and optionally a 3′ terminal region (translation and/or transcription terminator). The signal peptide encoding region is adjacent to the polynucleotide of the invention and placed in proper reading frame. The signal peptide-encoding region is homologous or heterologous to the DNA molecule encoding the mature polypeptide and is compatible with the secretion apparatus of the host used for expression. The open reading frame constituted by the DNA molecule of the invention, solely or together with the signal peptide, is placed under the control of the promoter so that transcription and translation occur in the host system. Promoters and signal peptide encoding regions are widely known and available to those skilled in the art and include, for example, the promoter of [0120] Salmonella typhimurium (and derivatives) that is inducible by arabinose (promoter araB) and is functional in Gram-negative bacteria such as E. coli (as described in U.S. Pat. No. 5,028,530 and in Cagnon et al., (Cagnon et al., Protein Engineering (1991) 4(7):843)); the promoter of the gene of bacteriophage T7 encoding RNA polymerase, that is functional in a number of E. coli strains expressing T7 polymerase (described in U.S. Pat. No. 4,952,496); OspA lipidation signal peptide; and RlpB lipidation signal peptide (Takase et al., J. Bact. (1987) 169:5692).
  • The expression cassette is typically part of an expression vector, which is selected for its ability to replicate in the chosen expression system. Expression vectors (e.g., plasmids or viral vectors) can be chosen, for example, from those described in Pouwels et al. (Cloning Vectors: A Laboratory Manual 1985, Supp. 1987). Suitable expression vectors can be purchased from various commercial sources. [0121]
  • Methods for transforming/transfecting host cells with expression vectors are well-known in the art and depend on the host system selected as described in Ausubel et al., (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994). [0122]
  • Upon expression, a recombinant polypeptide of the invention (or a polypeptide derivative) is produced and remains in the intracellular compartment, is secreted/excreted in the extracellular medium or in the periplasmic space, or is embedded in the cellular membrane. The polypeptide is recovered in a substantially purified form from the cell extract or from the supernatant after centrifugation of the recombinant cell culture. Typically, the recombinant polypeptide is purified by antibody-based affinity purification or by other well-known methods that can be readily adapted by a person skilled in the art, such as fusion of the polynucleotide encoding the polypeptide or its derivative to a small affinity binding domain. Antibodies useful for purifying by immunoaffinity the polypeptides of the invention are obtained as described below. [0123]
  • A polynucleotide of the invention can also be useful as a vaccine. There are two major routes, either using a viral or bacterial host as gene delivery vehicle (live vaccine vector) or administering the gene in a free form, e.g., inserted into a plasmid. Therapeutic or prophylactic efficacy of a polynucleotide of the invention is evaluated as described below. [0124]
  • Accordingly, a third aspect of the invention provides (i) a vaccine vector such as a poxvirus, containing a DNA molecule of the invention, placed under the control of elements required for expression; (ii) a composition of matter comprising a vaccine vector of the invention, together with a diluent or carrier; specifically (iii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a vaccine vector of the invention; (iv) a method for inducing an immune response against [0125] Chlamydia in a mammal (e.g., a human; alternatively, the method can be used in veterinary applications for treating or preventing Chlamydia infection of animals, e.g., cats or birds), which involves administering to the mammal an immunogenically effective amount of a vaccine vector of the invention to elicit a protective or therapeutic immune response to Chlamydia; and particularly, (v) a method for preventing and/or treating a Chlamydia (e.g., C. trachomatis, C. psittaci, C. pneumonia, C. pecorum) infection, which involves administering a prophylactic or therapeutic amount of a vaccine vector of the invention to an infected individual. Additionally, the third aspect of the invention encompasses the use of a vaccine vector of the invention in the preparation of a medicament for preventing and/or treating Chlamydia infection.
  • As used herein, a vaccine vector expresses one or several polypeptides or derivatives of the invention. The vaccine vector may express additionally a cytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12), that enhances the immune response (adjuvant effect). It is understood that each of the components to be expressed is placed under the control of elements required for expression in a mammalian cell. [0126]
  • Consistent with the third aspect of the invention is a composition comprising several vaccine vectors, each of them capable of expressing a polypeptide or derivative of the invention. A composition may also comprise a vaccine vector capable of expressing an additional [0127] Chlamydia antigen, or a subunit, fragment, homolog, mutant, or derivative thereof; optionally together with or a cytokine such as IL-2 or IL-12.
  • A general principle is that recognition of a particular antigen is not in itself sufficient to produce an effective immune response. In some cases, a cell-mediated response is appropriate; in others, antibody. [0128]
  • Antigens of microorganisms vary considerably in their accessibility to cells of the immune system. Antigens which normally occur inside a pathogen may become accessible only when the pathogen or an infected cell is killed. Even antigens expressed at the cell surface may present only a limited range of their potential epitopes for antibody binding, depending on their orientation in the membrane. Protective structures, such as bacterial capsules, further limit the effective recognition of epitopes. [0129]
  • A distinction should be drawn between the overall composition of the immune response, those components of it which are important in the resolution of infection and the components which are responsible for the prevention of re-infection. In many cases, particular elements of the immune response are critically important; for example, cell-mediated immunity in leprosy. Even when considering a particular effector system, the response directed against some antigens is often much more effective than the responses to others. Immune responses to particular microbial antigens have different degrees of relevance to anti-microbial immunity, depending on the nature of the organism, it pathogenicity and the nature of the immune response it initiates. [0130]
  • The primary effectors against extracellular pathogens are antibody and complement. Binding of antibody to receptors on the pathogen can prevent it from attaching to its target cell. Antibody alone, or more effectively in association with complement, opsonizes pathogens for uptake by phagocytes expressing Fc receptors and complement receptors CR1 and CR3. Usually this will lead to intracellular destruction of the pathogen but if the phagocyte is unable to destroy it and is a facultative host cell, then antibody may actually promote the spread of infection. Such an eventuality, however, depends on the dynamic balance between the actions of the humoral and cell-mediated immune responses. [0131]
  • Sometimes effective antibodies must be of the right class to activate appropriate effectors. The important antigens are those involved in evasion of immune effector mechanisms; that is, pili, fimbriae and capsular antigens which constitute the major antigens of the outer layer of bacteria. Often epitope specificity is important, since it determines whether complement is deposited in a position to damage the outer membrane. There are also numerous protein antigens which can induce an antibody response; however, although the antibody response is partly species-specific and may be diagnostically useful, it is largely irrelevant to immunity. This is most obvious in lepromatous leprosy, where the patients have weak cell-mediated immunity, high levels of specific antibody and tissues heavily infected with bacteria. [0132]
  • In some cases, a particular type of antibody response is mandatory for clearance of the pathogen. This is true of many bacterial infections, where specific antibodies to surface antigens are necessary to neutralize the bacterial defences and opsonize the bacteria for phagocytes. [0133]
  • There are also cases where responses to individual antigens are essential for host immunity. The simplest examples are the toxins produced by the causative agents of diphtheria, tetanus and clostridial enteritis. The damage produced directly by the infectious agent in these diseases is slight by comparison with that produced by the secreted toxins. Consequently, protection against these conditions involves immunization to toxoids. Nevertheless, the immune system must still eradicate the primary site of the bacterial infection if the disease is to be resolved. The target antigens for bactericidal antibodies are extremely diverse and include LPS, capsular polysaccharides and other outer membrane proteins. Virulence factors can also provide good immunogens in a vaccine. [0134]
  • Tables 1, 3, 4, 6, 8, 10, 12, 14, 16 and 18, as well as corresponding FIGS. [0135] 31 to 40, demonstrate that the polypeptides disclosed herein are immunogenic. Furthermore, these Figures demonstrate that the polypeptides disclosed herein confer immunoprotection from Chlamydia infection, as evidenced by accelerated clearance of pulmonary infection. Such reduction in the severity of effects of Chlamydia infection is evidence that the polypeptides have generated an active functional immune response against the pathogen, rather than a mere antibody response against the antigen.
  • Animal models have been used to define the immunobiologic feature of [0136] C. trachomatis infection. The mouse model is particularly informative, largely because of the ready availability of immune reagents for murine studies and the development of transgenic and knockout (KO) mice. C. trachomatis mouse pneumonitis (MoPn) is the most widely tested biovar among the three C. trachomatis biovars (trachoma, lymphogranuloma venereum, and MoPn). Although human biovars have also been used in animal models, they normally require high inocula or pretreatment with progesterone. MoPn, which was originally isolated from mouse tissues, is thought to be a natural murine pathogen and thus offers an evolutionarily adapted pathogen for analysis of host-pathogen interactions.
  • The significant progress in chlamydial immunobiology based on murine models of MoPn infection has extended and clarified recent immunoepidemiologic studies in humans (Yang and Brunham (1998) Can J Infect Dis; 9:99-108). In particular, since the discovery of T helper (Th) 1 and 2 subsets, cytokine patterns have been shown to be critical in the regulation of immune responses to a variety of infectious agents including chlamydiae. Clinical investigation has shown that trachoma patients with severe conjunctival scarring have impaired cell-mediated immune responses to [0137] C. trachomatis and high IgG antibody titers (Yang and Brunham (1999) Curr Opin Infect Dis; 12:47-52). Cytokine analysis shows increased interleukin (IL)-4 and reduced interferon (IFN)-γ production in subjects with scarring disease due to C. trachomatis infection compared with controls without scarring disease.
  • Vaccination methods for treating or preventing infection in a mammal comprises use of a vaccine vector of the invention to be administered by any conventional route, particularly to a mucosal (e.g., ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract) surface or via the parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route. Preferred routes depend upon the choice of the vaccine vector. Treatment may be effected in a single dose or repeated at intervals. The appropriate dosage depends on various parameters understood by skilled artisans such as the vaccine vector itself, the route of administration or the condition of the mammal to be vaccinated (weight, age and the like). [0138]
  • Live vaccine vectors available in the art include viral vectors such as adenoviruses and poxviruses as well as bacterial vectors, e.g., [0139] Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bilié de Calmette-Guérin (BCG), and Streptococcus.
  • An example of an adenovirus vector, as well as a method for constructing an adenovirus vector capable of expressing a DNA molecule of the invention, are described in U.S. Pat. No. 4,920,209. Poxvirus vectors include vaccinia and canary pox virus, described in U.S. Pat. No. 4,722,848 and U.S. Pat. No. 5,364,773, respectively. (Also see, e.g., Tartaglia et al., Virology (1992) 188:217) for a description of a vaccinia virus vector and Taylor et al, Vaccine (1995) 13:539 for a reference of a canary pox.) Poxvirus vectors capable of expressing a polynucleotide of the invention are obtained by homologous recombination as described in Kieny et al., Nature (1984) 312:163 so that the polynucleotide of the invention is inserted in the viral genome under appropriate conditions for expression in mammalian cells. Generally, the dose of vaccine viral vector, for therapeutic or prophylactic use, can be of from about 1×10[0140] 4 to about 1×1011, advantageously from about 1×107 to about 1×1010, preferably of from about 1×107 to about 1×109 plaque-forming units per kilogram. Preferably, viral vectors are administered parenterally; for example, in 3 doses, 4 weeks apart. It is preferable to avoid adding a chemical adjuvant to a composition containing a viral vector of the invention and thereby minimizing the immune response to the viral vector itself.
  • Non-toxicogenic [0141] Vibrio cholerae mutant strains that are useful as a live oral vaccine are known. Mekalanos et al., Nature (1983) 306:551 and U.S. Pat. No. 4,882,278 describe strains which have a substantial amount of the coding sequence of each of the two ctxA alleles deleted so that no functional cholerae toxin is produced. WO 92/11354 describes a strain in which the irgA locus is inactivated by mutation; this mutation can be combined in a single strain with ctxA mutations. WO 94/01533 describes a deletion mutant lacking functional ctxA and attRS1 DNA sequences. These mutant strains are genetically engineered to express heterologous antigens, as described in WO 94/19482. An effective vaccine dose of a Vibrio cholerae strain capable of expressing a polypeptide or polypeptide derivative encoded by a DNA molecule of the invention contains about 1×105 to about 1×109, preferably about 1×106 to about 1×108, viable bacteria in a volume appropriate for the selected route of administration. Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.
  • Attenuated [0142] Salmonella typhimurium strains, genetically engineered for recombinant expression of heterologous antigens or not, and their use as oral vaccines are described in Nakayama et al. (Bio/Technology (1988) 6:693) and WO 92/11361. Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.
  • Other bacterial strains used as vaccine vectors in the context of the present invention are described for [0143] Shigella flexneri in High et al., EMBO (1992) 11:1991 and Sizemore et al., Science (1995) 270:299; for Streptococcus gordonii in Medaglini et al., Proc. Natl. Acad. Sci. USA (1995) 92:6868; and for Bacille Calmette Guerin in Flynn J. L., Cell. Mol. Biol. (1994) 40 (suppl. I):31, WO 88/06626, WO 90/00594, WO 91/13157, WO 92/01796, and WO 92/21376.
  • In bacterial vectors, the polynucleotide of the invention is inserted into the bacterial genome or remains in a free state as part of a plasmid. [0144]
  • The composition comprising a vaccine bacterial vector of the present invention may further contain an adjuvant. A number of adjuvants are known to those skilled in the art. Preferred adjuvants are selected as provided below. [0145]
  • Accordingly, a fourth aspect of the invention provides (i) a composition of matter comprising a polynucleotide of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a polynucleotide of the invention; (iii) a method for inducing an immune response against [0146] Chlamydia in a mammal by administration of an immunogenically effective amount of a polynucleotide of the invention to elicit a protective immune response to Chlamydia; and particularly, (iv) a method for preventing and/or treating a Chlamydia (e.g., C. trachomatis, C. psittaci, C. pneumoniae, or C. pecorum) infection, by administering a prophylactic or therapeutic amount of a polynucleotide of the invention to an infected individual. Additionally, the fourth aspect of the invention encompasses the use of a polynucleotide of the invention in the preparation of a medicament for preventing and/or treating Chlamydia infection. A preferred use includes the use of a DNA molecule placed under conditions for expression in a mammalian cell, especially in a plasmid that is unable to replicate in mammalian cells and to substantially integrate in a mammalian genome.
  • Use of the polynucleotides of the invention include their administration to a mammal as a vaccine, for therapeutic or prophylactic purposes. Such polynucleotides are used in the form of DNA as part of a plasmid that is unable to replicate in a mammalian cell and unable to integrate into the mammalian genome. Typically, such a DNA molecule is placed under the control of a promoter suitable for expression in a mammalian cell. The promoter functions either ubiquitously or tissue-specifically. Examples of non-tissue specific promoters include the early Cytomegalovirus (CMV) promoter (described in U.S. Pat. No. 4,168,062) and the Rous Sarcoma Virus promoter (described in Norton & Coffin, Molec. Cell Biol. (1985) 5:281). An example of a tissue-specific promoter is the desmin promoter which drives expression in muscle cells (Li et al., Gene (1989) 78:243, Li & Paulin, J. Biol. Chem. (1991) 266:6562 and Li & Paulin, J. Biol. Chem. (1993) 268:10403). Use of promoters is well-known to those skilled in the art. Useful vectors are described in numerous publications, specifically WO 94/21797 and Hartikka et al., Human Gene Therapy (1996) 7:1205. [0147]
  • Polynucleotides of the invention which are used as vaccines encode either a precursor or a mature form of the corresponding polypeptide. In the precursor form, the signal peptide is either homologous or heterologous. In the latter case, a eucaryotic leader sequence such as the leader sequence of the tissue-type plasminogen factor (tPA) is preferred. [0148]
  • As used herein, a composition of the invention contains one or several polynucleotides with optionally at least one additional polynucleotide encoding another [0149] Chlamydia antigen such as urease subunit A, B, or both, or a fragment, derivative, mutant, or analog thereof. The composition may also contain an additional polynucleotide encoding a cytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12) so that the immune response is enhanced. These additional polynucleotides are placed under appropriate control for expression. Advantageously, DNA molecules of the invention and/or additional DNA molecules to be included in the same composition, are present in the same plasmid.
  • Standard techniques of molecular biology for preparing and purifying polynucleotides are used in the preparation of polynucleotide therapeutics of the invention. For use as a vaccine, a polynucleotide of the invention is formulated according to various methods outlined below. [0150]
  • One method utililizes the polynucleotide in a naked form, free of any delivery vehicles. Such a polynucleotide is simply diluted in a physiologically acceptable solution such as sterile saline or sterile buffered saline, with or without a carrier. When present, the carrier preferably is isotonic, hypotonic, or weakly hypertonic, and has a relatively low ionic strength, such as provided by a sucrose solution, e.g., a solution containing 20% sucrose. [0151]
  • An alternative method utilizes the polynucleotide in association with agents that assist in cellular uptake. Examples of such agents are (i) chemicals that modify cellular permeability, such as bupivacaine (see, e.g., WO 94/16737), (ii) liposomes for encapsulation of the polynucleotide, or (iii) cationic lipids or silica, gold, or tungsten microparticles which associate themselves with the polynucleotides. [0152]
  • Anionic and neutral liposomes are well-known in the art (see, e.g., Liposomes: A Practical Approach, RPC New Ed, IRL press (1990), for a detailed description of methods for making liposomes) and are useful for delivering a large range of products, including polynucleotides. [0153]
  • Cationic lipids are also known in the art and are commonly used for gene delivery. Such lipids include Lipofectin™ also known as DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (1,2-bis(oleyloxy)-3-(trimethylammonio)propane), DDAB (dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycyl spermine) and cholesterol derivatives such as DC-Chol (3 beta-(N-(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol). A description of these cationic lipids can be found in EP 187,702, WO 90/11092, U.S. Pat. No. 5,283,185, WO 91/15501, WO 95/26356, and U.S. Pat. No. 5,527,928. Cationic lipids for gene delivery are preferably used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine), as described in WO 90/11092 as an example. [0154]
  • Formulation containing cationic liposomes may optionally contain other transfection-facilitating compounds. A number of them are described in WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/02397. They include spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane (see, for example, WO 93/18759) and membrane-permeabilizing compounds such as GALA, Gramicidine S, and cationic bile salts (see, for example, WO 93/19768). [0155]
  • Gold or tungsten microparticles are used for gene delivery, as described in WO 91/00359, WO 93/17706, and Tang et al. Nature (1992) 356:152. The microparticle-coated polynucleotide is injected via intradermal or intraepidermal routes using a needleless injection device (“gene gun”), such as those described in U.S. Pat. No. 4,945,050, U.S. Pat. No. 5,015,580, and WO 94/24263. [0156]
  • The amount of DNA to be used in a vaccine recipient depends, e.g., on the strength of the promoter used in the DNA construct, the immunogenicity of the expressed gene product, the condition of the mammal intended for administration (e.g., the weight, age, and general health of the mammal), the mode of administration, and the type of formulation. In general, a therapeutically or prophylactically effective dose from about 1 μg to about 1 mg, preferably, from about 10 μg to about 800 μg and, more preferably, from about 25 μg to about 250 μg, can be administered to human adults. The administration can be achieved in a single dose or repeated at intervals. [0157]
  • The route of administration is any conventional route used in the vaccine field. As general guidance, a polynucleotide of the invention is administered via a mucosal surface, e.g., an ocular, intranasal, pulmonary, oral, intestinal, rectal, vaginal, and urinary tract surface; or via a parenteral route, e.g., by an intravenous, subcutaneous, intraperitoneal, intradermal, intraepidermal, or intramuscular route. The choice of administration route depends on the formulation that is selected. A polynucleotide formulated in association with bupivacaine is advantageously administered into muscles. When a neutral or anionic liposome or a cationic lipid, such as DOTMA or DC-Chol, is used, the formulation can be advantageously injected via intravenous, intranasal (aerosolization), intramuscular, intradermal, and subcutaneous routes. A polynucleotide in a naked form can advantageously be administered via the intramuscular, intradermal, or sub-cutaneous routes. [0158]
  • Although not absolutely required, such a composition can also contain an adjuvant. If so, a systemic adjuvant that does not require concomitant administration in order to exhibit an adjuvant effect is preferable such as, e.g., QS21, which is described in U.S. Pat. No. 5,057,546. [0159]
  • The sequence information provided in the present application enables the design of specific nucleotide probes and primers that are used for diagnostic purposes. Accordingly, a fifth aspect of the invention provides a nucleotide probe or primer having a sequence found in or derived by degeneracy of the genetic code from a sequence shown in any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. [0160]
  • The term “probe” as used in the present application refers to DNA (preferably single stranded) or RNA molecules (or modifications or combinations thereof) that hybridize under the stringent conditions, as defined above, to nucleic acid molecules having any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 or to a sequence homologous to any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, or to its complementary or anti-sense sequence. Generally, probes are significantly shorter than full-length sequences. Such probes contain from about 5 to about 100, preferably from about 10 to about 80, nucleotides. In particular, probes have sequences that are at least 75%, preferably at least 80% or 85%, more preferably 90% or 95% homologous to a portion of any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 or that are complementary to such sequences. Probes may contain modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, or diamino-2,6-purine. Sugar or phosphate residues may also be modified or substituted. For example, a deoxyribose residue may be replaced by a polyamide (Nielsen et al., Science (1991) 254:1497) and phosphate residues may be replaced by ester groups such as diphosphate, alkyl, arylphosphonate and phosphorothioate esters. In addition, the 2′-hydroxyl group on ribonucleotides may be modified by including such groups as alkyl groups. [0161]
  • Probes of the invention are used in diagnostic tests, as capture or detection probes. Such capture probes are conventionally immobilized on a solid support, directly or indirectly, by covalent means or by passive adsorption. A detection probe is labelled by a detection marker selected from: radioactive isotopes, enzymes such as peroxidase, alkaline phosphatase, and enzymes able to hydrolyze a chromogenic, fluorogenic, or luminescent substrate, compounds that are chromogenic, fluorogenic, or luminescent, nucleotide base analogs, and biotin. [0162]
  • Probes of the invention are used in any conventional hybridization technique, such as dot blot (Maniatis et al., Molecular Cloning: A Laboratory Manual (1982) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), Southern blot (Southern, J. Mol. Biol. (1975) 98:503), northern blot (identical to Southern blot with the exception that RNA is used as a target), or the sandwich technique (Dunn et al., Cell (1977) 12:23). The latter technique involves the use of a specific capture probe and/or a specific detection probe with nucleotide sequences that at least partially differ from each other. [0163]
  • A primer is a probe of usually about 10 to about 40 nucleotides that is used to initiate enzymatic polymerization of DNA in an amplification process (e.g., PCR), in an elongation process, or in a reverse transcription method. Primers used in diagnostic methods involving PCR are labeled by methods known in the art. [0164]
  • As described herein, the invention also encompasses (i) a reagent comprising a probe of the invention for detecting and/or identifying the presence of [0165] Chlamydia in a biological material; (ii) a method for detecting and/or identifying the presence of Chlamydia in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA or RNA is extracted from the material and denatured, and (c) exposed to a probe of the invention, for example, a capture, detection probe or both, under stringent hybridization conditions, such that hybridization is detected; and (iii) a method for detecting and/or identifying the presence of Chlamydia in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA is extracted therefrom, (c) the extracted DNA is primed with at least one, and preferably two, primers of the invention and amplified by polymerase chain reaction, and (d) the amplified DNA fragment is produced.
  • It is apparent that disclosure of a polynucleotide sequence of any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, its homologs and partial sequences enable their corresponding amino acid sequences. Accordingly, a sixth aspect of the invention features a substantially purified polypeptide or polypeptide derivative having an amino acid sequence encoded by a polynucleotide of the invention. [0166]
  • A “substantially purified polypeptide” as used herein is defined as a polypeptide that is separated from the environment in which it naturally occurs and/or that is free of the majority of the polypeptides that are present in the environment in which it was synthesized. For example, a substantially purified polypeptide is free from cytoplasmic polypeptides. Those skilled in the art would readily understand that the polypeptides of the invention may be purified from a natural source, i.e., a [0167] Chlamydia strain, or produced by recombinant means.
  • Consistent with the sixth aspect of the invention are polypeptides, homologs or fragments which are modified or treated to enhance their immunogenicity in the target animal, in whom the polypeptide, homolog or fragments are intended to confer protection against [0168] Chlamydia. Such modifications or treatments include: amino acid substitutions with an amino acid derivative such as 3-methyhistidine, 4-hydroxyproline, 5-hydroxylysine etc., modifications or deletions which are carried out after preparation of the polypeptide, homolog or fragment, such as the modification of free amino, carboxyl or hydroxyl side groups of the amino acids.
  • Identification of homologous polypeptides or polypeptide derivatives encoded by polynucleotides of the invention which have specific antigenicity is achieved by screening for cross-reactivity with an antiserum raised against the polypeptide of reference having an amino acid sequence of any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20. The procedure is as follows: a monospecific hyperimmune antiserum is raised against a purified reference polypeptide, a fusion polypeptide (for example, an expression product of MBP, GST, or His-tag systems, the description and instructions for use of which are contained in Invitrogen product manuals for pcDNA3.1/Myc-His(+) A, B, and C and for the Xpress™ System Protein Purification), or a synthetic peptide predicted to be antigenic. Where an antiserum is raised against a fusion polypeptide, two different fusion systems are employed. Specific antigenicity can be determined according to a number of methods, including Western blot (Towbin et al., Proc. Natl. Acad. Sci. USA (1979) 76:4350), dot blot, and ELISA, as described below. [0169]
  • In a Western blot assay, the product to be screened, either as a purified preparation or a total [0170] E. coli extract, is submitted to SDS-Page electrophoresis as described by Laemmli (Nature (1970) 227:680). After transfer to a nitrocellulose membrane, the material is further incubated with the monospecific hyperimmune antiserum diluted in the range of dilutions from about 1:5 to about 1:5000, preferably from about 1:100 to about 1:500. Specific antigenicity is shown once a band corresponding to the product exhibits reactivity at any of the dilutions in the above range.
  • In an ELISA assay, the product to be screened is preferably used as the coating antigen. A purified preparation is preferred, although a whole cell extract can also be used. Briefly, about 100 μl of a preparation at about 10 μg protein/ml are distributed into wells of a 96-well polycarbonate ELISA plate. The plate is incubated for 2 hours at 37° C. then overnight at 4° C. The plate is washed with phosphate buffer saline (PBS) containing 0.05% Tween 20 (PBS/Tween buffer). The wells are saturated with 250 μl PBS containing 1% bovine serum albumin (BSA) to prevent non-specific antibody binding. After 1 hour incubation at 37° C., the plate is washed with PBS/Tween buffer. The antiserum is serially diluted in PBS/Tween buffer containing 0.5% BSA. 100 μl of dilutions are added per well. The plate is incubated for 90 minutes at 37° C., washed and evaluated according to standard procedures. For example, a goat anti-rabbit peroxidase conjugate is added to the wells when specific antibodies were raised in rabbits. Incubation is carried out for 90 minutes at 37° C. and the plate is washed. The reaction is developed with the appropriate substrate and the reaction is measured by colorimetry (absorbance measured spectrophotometrically). Under the above experimental conditions, a positive reaction is shown by O.D. values greater than a non immune control serum. [0171]
  • In a dot blot assay, a purified product is preferred, although a whole cell extract can also be used. Briefly, a solution of the product at about 100 μg/ml is serially two-fold diluted in 50 mM Tris-HCl (pH 7.5). 100 μl of each dilution are applied to a nitrocellulose membrane 0.45 μm set in a 96-well dot blot apparatus (Biorad). The buffer is removed by applying vacuum to the system. Wells are washed by addition of 50 mM Tris-HCl (pH 7.5) and the membrane is air-dried. The membrane is saturated in blocking buffer (50 mM Tris-HCl (pH 7.5) 0.15 M NaCl, 10 g/L skim milk) and incubated with an antiserum dilution from about 1:50 to about 1:5000, preferably about 1:500. The reaction is revealed according to standard procedures. For example, a goat anti-rabbit peroxidase conjugate is added to the wells when rabbit antibodies are used. Incubation is carried out 90 minutes at 37° C. and the blot is washed. The reaction is developed with the appropriate substrate and stopped. The reaction is measured visually by the appearance of a colored spot, e.g., by colorimetry. Under the above experimental conditions, a positive reaction is shown once a colored spot is associated with a dilution of at least about 1:5, preferably of at least about 1:500. [0172]
  • Therapeutic or prophylactic efficacy of a polypeptide or derivative of the invention can be evaluated as described below. A seventh aspect of the invention provides (i) a composition of matter comprising a polypeptide of the invention together with a diluent or carrier; specifically (ii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a polypeptide of the invention; (iii) a method for inducing an immune response against [0173] Chlamydia in a mammal, by administering to the mammal an immunogenically effective amount of a polypeptide of the invention to elicit a protective immune response to Chlamydia; and particularly, (iv) a method for preventing and/or treating a Chlamydia (e.g., C. trachomatis. C. psittaci, C. pneumoniae. or C. pecorum) infection, by administering a prophylactic or therapeutic amount of a polypeptide of the invention to an infected individual. Additionally, the seventh aspect of the invention encompasses the use of a polypeptide of the invention in the preparation of a medicament for preventing and/or treating Chlamydia infection.
  • As used herein, the immunogenic compositions of the invention are administered by conventional routes known the vaccine field, in particular to a mucosal (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface or via the parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route. The choice of administration route depends upon a number of parameters, such as the adjuvant associated with the polypeptide. If a mucosal adjuvant is used, the intranasal or oral route is preferred. If a lipid formulation or an aluminum compound is used, the parenteral route is preferred with the sub-cutaneous or intramuscular route being most preferred. The choice also depends upon the nature of the vaccine agent. For example, a polypeptide of the invention fused to CTB or LTB is best administered to a mucosal surface. [0174]
  • As used herein, the composition of the invention contains one or several polypeptides or derivatives of the invention. The composition optionally contains at least one additional [0175] Chlamydia antigen, or a subunit, fragment, homolog, mutant, or derivative thereof.
  • For use in a composition of the invention, a polypeptide or derivative thereof is formulated into or with liposomes, preferably neutral or anionic liposomes, microspheres, ISCOMS, or virus-like-particles (VLPs) to facilitate delivery and/or enhance the immune response. These compounds are readily available to one skilled in the art; for example, see Liposomes: A Practical Approach, RCP New Ed, IRL press (1990). [0176]
  • Adjuvants other than liposomes and the like are also used and are known in the art. Adjuvants may protect the antigen from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. An appropriate selection can conventionally be made by those skilled in the art, for example, from those described below (under the eleventh aspect of the invention). [0177]
  • Treatment is achieved in a single dose or repeated as necessary at intervals, as can be determined readily by one skilled in the art. For example, a priming dose is followed by three booster doses at weekly or monthly intervals. An appropriate dose depends on various parameters including the recipient (e.g., adult or infant), the particular vaccine antigen, the route and frequency of administration, the presence/absence or type of adjuvant, and the desired effect (e.g., protection and/or treatment), as can be determined by one skilled in the art. In general, a vaccine antigen of the invention is administered by a mucosal route in an amount from about 10 μg to about 500 mg, preferably from about 1 mg to about 200 mg. For the parenteral route of administration, the dose usually does not exceed about 1 mg, preferably about 100 μg. [0178]
  • When used as vaccine agents, polynucleotides and polypeptides of the invention may be used sequentially as part of a multistep immunization process. For example, a mammal is initially primed with a vaccine vector of the invention such as a pox virus, e.g., via the parenteral route, and then boosted twice with the polypeptide encoded by the vaccine vector, e.g., via the mucosal route. In another example, liposomes associated with a polypeptide or derivative of the invention is also used for priming, with boosting being carried out mucosally using a soluble polypeptide or derivative of the invention in combination with a mucosal adjuvant (e.g., LT). [0179]
  • A polypeptide derivative of the invention is also used in accordance with the seventh aspect as a diagnostic reagent for detecting the presence of anti-[0180] Chlamydia antibodies, e.g., in a blood sample. Such polypeptides are about 5 to about 80, preferably about 10 to about 50 amino acids in length. They are either labeled or unlabeled, depending upon the diagnostic method. Diagnostic methods involving such a reagent are described below.
  • Upon expression of a DNA molecule of the invention, a polypeptide or polypeptide derivative is produced and purified using known laboratory techniques. As described above, the polypeptide or polypeptide derivative may be produced as a fusion protein containing a fused tail that facilitates purification. The fusion product is used to immunize a small mammal, e.g., a mouse or a rabbit, in order to raise antibodies against the polypeptide or polypeptide derivative (monospecific antibodies). Accordingly, an eighth aspect of the invention provides a monospecific antibody that binds to a polypeptide or polypeptide derivative of the invention. [0181]
  • By “monospecific antibody” is meant an antibody that is capable of reacting with a unique naturally-occurring [0182] Chlamydia polypeptide. An antibody of the invention is either polyclonal or monoclonal. Monospecific antibodies may be recombinant, e.g., chimeric (e.g., constituted by a variable region of murine origin associated with a human constant region), humanized (a human immunoglobulin constant backbone together with hypervariable region of animal, e.g., murine, origin), and/or single chain. Both polyclonal and monospecific antibodies may also be in the form of immunoglobulin fragments, e.g., F(ab)′2 or Fab fragments. The antibodies of the invention are of any isotype, e.g., IgG or IgA, and polyclonal antibodies are of a single isotype or a mixture of isotypes.
  • Antibodies against the polypeptides, homologs or fragments of the present invention are generated by immunization of a mammal with a composition comprising said polypeptide, homolog or fragment. Such antibodies may be polyclonal or monoclonal. Methods to produce polyclonal or monoclonal antibodies are well known in the art. For a review, see “Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Eds. E. Harlow and D. Lane (1988), and D. E. Yelton et al., 1981. Ann. Rev. Biochem. 50:657-680. For monoclonal antibodies, see Kohler & Milstein (1975) Nature 256:495-497. [0183]
  • The antibodies of the invention, which are raised to a polypeptide or polypeptide derivative of the invention, are produced and identified using standard immunological assays, e.g., Western blot analysis, dot blot assay, or ELISA (see, e.g., Coligan et al., Current Protocols in Immunology (1994) John Wiley & Sons, Inc., New York, N.Y.). The antibodies are used in diagnostic methods to detect the presence of a [0184] Chlamydia antigen in a sample, such as a biological sample. The antibodies are also used in affinity chromatography for purifying a polypeptide or polypeptide derivative of the invention. As is discussed further below, such antibodies may be used in prophylactic and therapeutic passive immunization methods.
  • Accordingly, a ninth aspect of the invention provides (i) a reagent for detecting the presence of [0185] Chlamydia in a biological sample that contains an antibody, polypeptide, or polypeptide derivative of the invention; and (ii) a diagnostic method for detecting the presence of Chlamydia in a biological sample, by contacting the biological sample with an antibody, a polypeptide, or a polypeptide derivative of the invention, such that an immune complex is formed, and by detecting such complex to indicate the presence of Chlamydia in the sample or the organism from which the sample is derived.
  • Those skilled in the art will readily understand that the immune complex is formed between a component of the sample and the antibody, polypeptide, or polypeptide derivative, whichever is used, and that any unbound material is removed prior to detecting the complex. It is understood that a polypeptide reagent is useful for detecting the presence of anti-[0186] Chlamydia antibodies in a sample, e.g., a blood sample, while an antibody of the invention is used for screening a sample, such as a gastric extract or biopsy, for the presence of Chlamydia polypeptides.
  • For diagnostic applications, the reagent (i.e., the antibody, polypeptide, or polypeptide derivative of the invention) is either in a free state or immobilized on a solid support, such as a tube, a bead, or any other conventional support used in the field. Immobilization is achieved using direct or indirect means. Direct means include passive adsorption (non-covalent binding) or covalent binding between the support and the reagent. By “indirect means” is meant that an anti-reagent compound that interacts with a reagent is first attached to the solid support. For example, if a polypeptide reagent is used, an antibody that binds to it can serve as an anti-reagent, provided that it binds to an epitope that is not involved in the recognition of antibodies in biological samples. Indirect means may also employ a ligand-receptor system, for example, where a molecule such as a vitamin is grafted onto the polypeptide reagent and the corresponding receptor immobilized on the solid phase. This is illustrated by the biotin-streptavidin system. Alternatively, a peptide tail is added chemically or by genetic engineering to the reagent and the grafted or fused product immobilized by passive adsorption or covalent linkage of the peptide tail. [0187]
  • Such diagnostic agents may be included in a kit which also comprises instructions for use. The reagent is labeled with a detection means which allows for the detection of the reagent when it is bound to its target. The detection means may be a fluorescent agent such as fluorescein isocyanate or fluorescein isothiocyanate, or an enzyme such as horse radish peroxidase or luciferase or alkaline phosphatase, or a radioactive element such as [0188] 125I, or 51Cr.
  • Accordingly, a tenth aspect of the invention provides a process for purifying, from a biological sample, a polypeptide or polypeptide derivative of the invention, which involves carrying out antibody-based affinity chromatography with the biological sample, wherein the antibody is a monospecific antibody of the invention. [0189]
  • For use in a purification process of the invention, the antibody is either polyclonal or monospecific, and preferably is of the IgG type. Purified IgGs is prepared from an antiserum using standard methods (see, e.g., Coligan et al., Current Protocols in Immunology (1994)John Wiley & Sons, Inc., New York, N.Y.). Conventional chromatography supports, as well as standard methods for grafting antibodies, are described in, e.g., Antibodies: A Laboratory Manual, D. Lane, E. Harlow, Eds. (1988) and outlined below. [0190]
  • Briefly, a biological sample, such as an [0191] C. pneumoniae extract preferably in a buffer solution, is applied to a chromatography material, preferably equilibrated with the buffer used to dilute the biological sample so that the polypeptide or polypeptide derivative of the invention (i.e., the antigen) is allowed to adsorb onto the material. The chromatography material, such as a gel or a resin coupled to an antibody of the invention, is in either a batch form or a column. The unbound components are washed off and the antigen is then eluted with an appropriate elution buffer, such as a glycine buffer or a buffer containing a chaotropic agent, e.g., guanidine HCl, or high salt concentration (e.g., 3 M MgCl2). Eluted fractions are recovered and the presence of the antigen is detected, e.g., by measuring the absorbance at 280 nm.
  • An eleventh aspect of the invention provides (i) a composition of matter comprising a monospecific antibody of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a monospecific antibody of the invention, and (iii) a method for treating or preventing a [0192] Chlamydia (e.g., C. trachomatis, C. psittaci, C. pneumoniae or C. pecorum) infection, by administering a therapeutic or prophylactic amount of a monospecific antibody of the invention to an infected individual. Additionally, the eleventh aspect of the invention encompasses the use of a monospecific antibody of the invention in the preparation of a medicament for treating or preventing Chlamydia infection.
  • The monospecific antibody is either polyclonal or monoclonal, preferably of the IgA isotype (predominantly). In passive immunization, the antibody is administered to a mucosal surface of a mammal, e.g., the gastric mucosa, e.g., orally or intragastrically, advantageously, in the presence of a bicarbonate buffer. Alternatively, systemic administration, not requiring a bicarbonate buffer, is carried out. A monospecific antibody of the invention is administered as a single active component or as a mixture with at least one monospecific antibody specific for a different [0193] Chlamydia polypeptide. The amount of antibody and the particular regimen used are readily determined by one skilled in the art. For example, daily administration of about 100 to 1,000 mg of antibodies over one week, or three doses per day of about 100 to 1,000 mg of antibodies over two or three days, are effective regimens for most purposes.
  • Therapeutic or prophylactic efficacy are evaluated using standard methods in the art, e.g., by measuring induction of a mucosal immune response or induction of protective and/or therapeutic immunity, using, e.g., the [0194] C. pneumoniae mouse model. Those skilled in the art will readily recognize that the C. pneumoniae strain of the model may be replaced with another Chlamydia strain. For example, the efficacy of DNA molecules and polypeptides from C. pneumoniae is preferably evaluated in a mouse model using C. pneumoniae strain. Protection is determined by comparing the degree of Chlamydia infection to that of a control group. Protection is shown when infection is reduced by comparison to the control group. Such an evaluation is made for polynucleotides, vaccine vectors, polypeptides and derivatives thereof, as well as antibodies of the invention.
  • Adjuvants useful in any of the vaccine compositions described above are as follows. [0195]
  • Adjuvants for parenteral administration include aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate. The antigen is precipitated with, or adsorbed onto, the aluminum compound according to standard protocols. Other adjuvants, such as RIBI (ImmunoChem, Hamilton, Mont.), are used in parenteral administration. [0196]
  • Adjuvants for mucosal administration include bacterial toxins, e.g., the cholera toxin (CT), the [0197] E. coli heat-labile toxin (LT), the Clostridium difficile toxin A and the pertussis toxin (PT), or combinations, subunits, toxoids, or mutants thereof such as a purified preparation of native cholera toxin subunit B (CTB). Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity. Preferably, a mutant having reduced toxicity is used. Suitable mutants are described, e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/06627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant). Additional LT mutants that are used in the methods and compositions of the invention include, e.g., Ser-63-Lys, Ala-69Gly, Glu-110-Asp, and Glu-112-Asp mutants. Other adjuvants, such as a bacterial monophosphoryl lipid A (MPLA) of, e.g., E. coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri; saponins, or polylactide glycolide (PLGA) microspheres, is also be used in mucosal administration.
  • Adjuvants useful for both mucosal and parenteral administrations include polyphosphazene (WO 95/02415), DC-chol (3 b-(N-(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol; U.S. Pat. No. 5,283,185 and WO 96/14831) and QS-21 (WO 88/09336). [0198]
  • Any pharmaceutical composition of the invention containing a polynucleotide, a polypeptide, a polypeptide derivative, or an antibody of the invention, is manufactured in a conventional manner. In particular, it is formulated with a pharmaceutically acceptable diluent or carrier, e.g., water or a saline solution such as phosphate buffer saline. In general, a diluent or carrier is selected on the basis of the mode and route of administration, and standard pharmaceutical practice. Suitable pharmaceutical carriers or diluents, as well as pharmaceutical necessities for their use in pharmaceutical formulations, are described in [0199] Remington's Pharmaceutical Sciences, a standard reference text in this field and in the USP/NF.
  • The invention also includes methods in which [0200] Chlamydia infection are treated by oral administration of a Chlamydia polypeptide of the invention and a mucosal adjuvant, in combination with an antibiotic, an antacid, sucralfate, or a combination thereof. Examples of such compounds that can be administered with the vaccine antigen and the adjuvant are antibiotics, including, e.g., macrolides, tetracyclines, and derivatives thereof (specific examples of antibiotics that can be used include azithromycin or doxicyclin or immunomodulators such as cytokines or steroids). In addition, compounds containing more than one of the above-listed components coupled together, are used. The invention also includes compositions for carrying out these methods, i.e., compositions containing a Chlamydia antigen (or antigens) of the invention, an adjuvant, and one or more of the above-listed compounds, in a pharmaceutically acceptable carrier or diluent.
  • It has recently been shown that the 60 kDa cysteine rich membrane protein contains a sequence cross-reactive with the murine alpha-myosin heavy chain epitope M7A-alpha, an epitope conserved in humans (Bachmaier et al., Science (1999) 283:1335). This cross-reactivity is proposed to contribute to the development of cardiovascular disease, so it may be beneficial to remove this epitope, and any other epitopes cross-reactive with human antigens, from the protein if it is to be used as a vaccine. Accordingly, a further embodiment of the present invention includes the modification of the coding sequence, for example, by deletion or substitution of the nucleotides encoding the epitope from polynucleotides encoding the protein, as to improve the efficacy and safety of the protein as a vaccine. A similar approach may be appropriate for any protective antigen found to have unwanted homologies or cross-reactivities with human antigens. [0201]
  • Amounts of the above-listed compounds used in the methods and compositions of the invention are readily determined by one skilled in the art. Treatment/immunization schedules are also known and readily designed by one skilled in the art. For example, the non-vaccine components can be administered on days 1-14, and the vaccine antigen+adjuvant can be administered on days 7, 14, 21, and 28. [0202]
    • EXAMPLES
  • The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation. [0203]
    • Example 1
  • These examples illustrate the preparation of plasmid vectors used in immunoprotection studies. [0204]
  • A. Preparation of Plasmid Vector pCACPNM213 [0205]
  • The ATP-binding cassette gene was amplified from [0206] Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAATGCGGCCGCCACCATGAAGATGCATAGGCTTAAACC 3′; SEQ ID No:21) and a 3′ primer (5′ GCGCCGGATCCCACTTAAGATATCGATATTTTTGAG 3′; SEQ ID No:22). The 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the ATP-binding cassette protein coding sequence. The 3′ primer includes the sequence encoding the C-terminal sequence of the ATP-binding cassette protein gene and a BamHI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • After amplification, the PCR fragment was purified using QIAquick™ PCR purification kit (Qiagen), digested with NotI and BamHI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 21) with transcription under control of the human CMV promoter. [0207]
  • B. Preparation of Plasmid Vector pCACPNM882 [0208]
  • The secretory locus ORF gene was amplified from [0209] Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAATGCGGCCGCCACCATGCGGTTGGGAAATAAGCCTATGC 3′; SEQ ID No:23) and a 3′ primer (5′ GCGCCGGTACCGTAATTTAATACTCTTTGAAGGGC 3′; SEQ ID No:24). The 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the secretory locus ORF coding sequence. The 3′ primer includes the sequence encoding the C-terminal sequence of the secretory locus ORF protein and a KpnI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • After amplification, the PCR fragment was purified using QIAquick™ PCR purification kit (Qiagen), digested with NotI and KpnI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 22) with transcription under control of the human CMV promoter. [0210]
  • C. Preparation of Plasmid Vector pCACPNM208 [0211]
  • The endopeptidase gene was amplified from [0212] Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAATGCGGCCGCCACCATGCTCACCCTAGGCTTGGAAAGTTCTTG 3′; SEQ ID No:25) and a 3′ primer (5′ GCTTTGGAGGATCCCCGGAGAGGCTAAGGAGAATGG 3′; SEQ ID No:26). The 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the endopeptidase protein coding sequence. The 3′ primer includes the sequence encoding the C-terminal sequence of the endopeptidase protein gene and a BamHI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • After amplification, the PCR fragment was purified using QIAquick™ PCR purification kit (Qiagen), digested with NotI and BamHI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 23) with transcription under control of the human CMV promoter. [0213]
  • D. Preparation of Plasmid Vector pCACPNM1096 [0214]
  • The protease gene was amplified from [0215] Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAATGCGGCCGCCACCATGAAAAAAGGGAAATTAGGAGCC 3′; SEQ ID No:27) and a 3′ primer (5′ GCGCCGGATCCCCGAAGCAGAAGTCGTTGTGGG 3′; SEQ ID No:28). The 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the protease protein coding sequence. The 3′ primer includes the sequence encoding the C-terminal sequence of the protease protein gene and a BamHI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • After amplification, the PCR fragment was purified using QIAquick™ PCR purification kit (Qiagen), digested with NotI and BamHI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 24) with transcription under control of the human CMV promoter. [0216]
  • E. Preparation of Plasmid Vector pCACPNM1097 [0217]
  • The metalloprotease gene was amplified from [0218] Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAATGCGGCCGCCACCATGAGAAAACTTATTTTATGCAATCCTA 3′; SEQ ID No:29) and a 3′ primer (5′ GCGCCGGATCCCAGAACAACGGAGTTCTTTTGG 3′; SEQ ID No:30). The 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the metalloprotease protein coding sequence. The 3′ primer includes the sequence encoding the C-terminal sequence of the metalloprotease protein gene and a BamHI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • After amplification, the PCR fragment was purified using QIAquick™ PCR purification kit (Qiagen), digested with NotI and BamHI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 25) with transcription under control of the human CMV promoter. [0219]
  • F. Preparation of Plasmid Vector pCACPNM908 [0220]
  • The CLP protease ATPase gene was amplified from [0221] Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAATGCGGCCGCCACCATGAATAAAAAAAATCTAACTATTTG 3′; SEQ ID No:31) and a 3′ primer (5′ GCGCCGGATCCCAGCGATAGCTTCTGGGGTCC 3′; SEQ ID No:32). The 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the CLP protease ATPase protein coding sequence. The 3′ primer includes the sequence encoding the C-terminal sequence of the CLP protease ATPase gene and a BamHI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • After amplification, the PCR fragment was purified using QIAquick™ PCR purification kit (Qiagen), digested with NotI and BamHI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 26) with transcription under control of the human CMV promoter. [0222]
  • G. Preparation of Plasmid Vector pCACPNM909 [0223]
  • The gene encoding CLP protease subunit was amplified from [0224] Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAATGCGGCCGCCACCATGACACTGGTACCCTATGTTG 3′; SEQ ID No:33) and a 3′ primer (5′ GCGCCGGATCCCAGTGCTACTTGTATCCTTATTAG 3′; SEQ ID No:34). The 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the CLP protease subunit coding sequence. The 3′ primer includes the sequence encoding the C-terminal sequence of the CLP protease subunit gene and a BamHI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • After amplification, the PCR fragment was purified using QIAquick™ PCR purification kit (Qiagen), digested with NotI and BamHI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 27) with transcription under control of the human CMV promoter. [0225]
  • H. Preparation of Plasmid Vector pCACPNM440 [0226]
  • The translycolase/transpeptidase gene was amplified from [0227] Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAATGCGGCCGCCACCATGAGCTACCGTAAACGTTCGACTC 3′; SEQ ID No:35) and a 3′ primer (5′ GCGCCGGATCCCCCTCGTTCCCCCTTGTTTCGGAG 3′; SEQ ID No:36). The 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the translycolase/transpeptidase coding sequence. The 3′ primer includes the sequence encoding the C-terminal sequence of the translycolase/transpeptidase gene and a BamHI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • After amplification, the PCR fragment was purified using QIAquick™ PCR purification kit (Qiagen), digested with NotI and BamHI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 28) with transcription under control of the human CMV promoter. [0228]
  • I. Preparation of Plasmid Vector pCACPNM459 [0229]
  • The gene encoding CLPc protease was amplified from [0230] Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAATGCGGCCGCCACCATGTTTGAGAAGTTCACTAATAGAGC 3′; SEQ ID No:37) and a 3′ primer (5′ GCGCCGGTACCGTGATTCCAAGTGAGGGCTAGGG 3′; SEQ ID No:38). The 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the CLPc protease coding sequence. The 3′ primer includes the sequence encoding the C-terminal sequence of the CLPc protease gene and a KpnI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • After amplification, the PCR fragment was purified using QIAquick™ PCR purification kit (Qiagen), digested with NotI and KpnI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 29) with transcription under control of the human CMV promoter. [0231]
  • J. Preparation of Plasmid Vector pCACPNM708 [0232]
  • The thioredoxin gene was amplified from [0233] Chlamydia pneumoniae genomic DNA strain CWLO29 by polymerase chain reaction (PCR) using a 5′ primer (5′ ATAAGAATGCGGCCGCCACCATGGTAAAGATCATATCAAGTG 3′; SEQ ID No:39) and a 3′ primer (5′ GCGCCGGATCCCAGCGTGCTTATTGATAAG 3′; SEQ ID No:40). The 5′ primer contains a NotI restriction site, a ribosome binding site, an initiation codon and a sequence at the 5′ end of the thioredoxin coding sequence. The 3′ primer includes the sequence encoding the C-terminal sequence of the thioredoxin gene and a BamHI restriction site. The stop codon was excluded and an additional nucleotide was inserted to obtain an in-frame fusion with the Histidine tag.
  • After amplification, the PCR fragment was purified using QIAquick™ PCR purification kit (Qiagen), digested with NotI and BamHI and cloned into the pCA-Myc-His eukaryotic expression vector described in Example 2 (FIG. 30) with transcription under control of the human CMV promoter. [0234]
    • Example 2
  • Plasmid pcDNA3.1(−)Myc-His C (Invitrogen) was restricted with SpeI and BamHI to remove the CMV promoter and the remaining vector fragment was isolated. The CMV promoter and intron A from plasmid VR-1012 (Vical) was isolated on a SpeI/BamHI fragment. The fragments were ligated together to produce plasmid pCA/Myc-His. [0235]
  • The NotI/BamHI restricted PCR fragment containing the ATP-binding cassette gene was ligated into the NotI and BamHI restricted plasmid pCA/Myc-His to produce plasmid pCACPNM213 (FIG. 21). [0236]
  • The NotI/KpnI restricted PCR fragment containing the Secretory locus ORF gene was ligated into the NotI and KpnI restricted plasmid pCA/Myc-His to produce plasmid pCACPNM882 (FIG. 22). [0237]
  • The NotI/BamHI restricted PCR fragment containing the endopeptidase gene was ligated into the NotI and BamHI restricted plasmid pCA/Myc-His to produce plasmid pCACPNM208 (FIG. 23). [0238]
  • The NotI/BamHI restricted PCR fragment containing the Protease gene was ligated into the NotI and BamHI restricted plasmid pCA/Myc-His to produce plasmid pCACPNM1096 (FIG. 24). [0239]
  • The NotI/BamHI restricted PCR fragment containing the Metalloprotease gene was ligated into the NotI and BamHI restricted plasmid pCA/Myc-His to produce plasmid pCACPNM1097 (FIG. 25). [0240]
  • The NotI/BamHI restricted PCR fragment containing the CLP protease ATPase gene was ligated into the NotI and BamHI restricted plasmid pCA/Myc-His to produce plasmid pCACPNM908 (FIG. 26). [0241]
  • The NotI/BamHI restricted PCR fragment containing the CLP protease subunit gene was ligated into the NotI and BamHI restricted plasmid pCA/Myc-His to produce plasmid pCACPNM909 (FIG. 27). [0242]
  • The NotI/BamHI restricted PCR fragment containing the transglycolase/transpeptidase gene was ligated into the NotI and BamHI restricted plasmid pCA/Myc-His to produce plasmid pCACPNM440 (FIG. 28). [0243]
  • The NotI/KpnI restricted PCR fragment containing the CLPc protease gene was ligated into the NotI and KpnI restricted plasmid pCA/Myc-His to produce plasmid pCACPNM459 (FIG. 29). [0244]
  • The NotI/BamHI restricted PCR fragment containing the Thioredoxin gene was ligated into the NotI and BamHI restricted plasmid pCA/Myc-His to produce plasmid pCACPNM708 (FIG. 30). [0245]
  • Each of the resulting plasmids pCACPNM213, pCACPNM882, pCACPNM208, pCACPNM1096, pCACPNM1097, pCACPNM909, pCACPNM440, pCACPNM459 and pCACPNM708, was transferred by electroporation into [0246] E. coli XL-1 blue (Stratagene) which was grown in LB broth containing 50 μg/ml carbenicillin. The plasmid was isolated by the Endo Free Plasmid Giga Kit™ (Qiagen) large scale DNA purification system. DNA concentration was determined by absorbance at 260 nm and the plasmid was verified after gel electrophoresis and ethidium bromide staining by comparison to molecular weight standards. The 5′ and 3′ ends of the gene were verified by sequencing using a LiCor model 4000 L DNA sequencer and IRD-800 labelled primers.
    • Example 3
  • This example illustrates the immunization of mice to achieve protection against an intranasal challenge of [0247] C. pneumoniae.
  • It has been previously demonstrated (Yang et al. Infect. Immun. May 1993. 61(5):2037-40) that mice are susceptible to intranasal infection with different isolates of [0248] C. pneumoniae. Strain AR-39 (Grayston et al (1990) Journal of Infectious Diseases 161:618-625) was used in Balb/c mice as a challenge infection model to examine the capacity of Chlamydia gene products delivered as naked DNA to elicit a protective response against a sublethal C. pneumoniae lung infection. Protective immunity is defined as an accelerated clearance of pulmonary infection.
  • Groups of 7 to 9 week old male Balb/c mice (8 to 10 per group) were immunized intramuscularly (i.m.) plus intranasally (i.n.) with plasmid DNA containing each of the [0249] C. pneumoniae protein gene as described in Examples 1 and 2. Saline or the plasmid vector lacking an inserted Chlamydial gene was given to groups of control animals.
  • For i.m. immunization, alternate left and right quadriceps were injected with 100 μg of DNA in 50 μl of PBS on three occasions at 0, 3 and 6 weeks. For i.n. immunization, anaesthetized mice were aspirated 50 μl of PBS containing 50 μg DNA on three occasions at 0, 3 and 6 weeks. At [0250] week 8, immunized mice were inoculated i.n. with 5×105 IFU of C. pneumoniae, strain AR39 in 100 μl of SPG buffer to test their ability to limit the growth of a sublethal C. pneumoniae challenge.
  • Lungs were taken from mice at [0251] day 9 post-challenge and immediately homogenised in SPG buffer (7.5% sucrose, 5mM glutamate, 12.5 mM phosphate pH7.5). The homogenate was stored frozen at −70° C. until assay. Dilutions of the homogenate were assayed for the presence of infectious Chlamydia by inoculation onto monolayers of susceptible cells. The inoculum was centrifuged onto the cells at 3000 rpm for 1 hour, then the cells were incubated for three days at 35° C. in the presence of 1 μg/ml cycloheximide. After incubation the monolayers were fixed with formalin and methanol then immunoperoxidase stained for the presence of Chlamydial inclusions using convalescent sera from rabbits infected with C. pneumoniae and metal-enhanced DAB as a peroxidase substrate.
  • A. Immunization with pCACPNM213 [0252]
  • FIG. 31 and Table 1 show that mice immunized i.n. and i.m. with pCACPNM213 had chlamydial lung titers less than 60,000 in 3 of 6 cases at day 9 (mean 51,833) whereas the range of values for control mice sham immunized with saline was 34,200-377,800 IFU/lung (mean 141,450) at [0253] day 9. DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM102, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 153,283). The construct pCACPNM102 is identical to pCACPNM213 except that the nucleotide sequence encoding the putative ATP-binding cassette is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated ATP Synthase Subunit I protein.
  • B. Immunization with pCACPNM882 [0254]
  • FIG. 32 and Table 3 show that mice immunized i.n. and i.m. with pCACPNM882 had chlamydial lung titers less than 73,000 in 4 of 6 cases at day 9 (mean 77,500) whereas the range of values for control mice sham immunized with saline was 56,000-424,000 IFU/lung (mean 186,291) at [0255] day 9. DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM647, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 143,883). The construct pCACPNM647 is identical to pCACPNM882 except that the nucleotide sequence encoding the putative Secretory locus ORF is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated substrate binding protein.
  • C. Immunization with pCACPNM208 [0256]
  • FIG. 33 and Table 4 show that mice immunized i.n. and i.m. with pCACPNM208 had chlamydial lung titers less than 67,000 in 4 of 6 cases at day 9 (mean 81,766) whereas the range of values for control mice sham immunized with saline was 56,000-424,100 IFU/lung (mean 186,291) at [0257] day 9. DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM647, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 143,883). The construct pCACPNM647 is identical to pCACPNM208 except that the nucleotide sequence encoding the putative Endopeptidase is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated protein.
    • D. Immunization with pCACPNM1096
  • FIG. 34 and Table 6 show that mice immunized i.n. and i.m. with pCACPNM1096 had chlamydial lung titers less than 30,000 in 5 of 6 cases at day 9 (mean 25,000) whereas the range of values for control mice sham immunized with saline was 51,300-170,000 IFU/lung (mean 105,150) at [0258] day 9. DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM553, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 111,583). The construct pCACPNM553 is identical to pCACPNM1096 except that the nucleotide sequence encoding the putative Protease is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated protease.
  • E. Immunization with pCACPNM1097 [0259]
  • FIG. 35 and Table 8 show that mice immunized i.n. and i.m. with pCACPNM1097 had chlamydial lung titers less than 51,000 in 4 of 6 cases at day 9 (mean 62,883) whereas the range of values for control mice sham immunized with saline was 90,000-242,100 IFU/lung (mean 166,287) at [0260] day 9. DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM1061, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 148,566). The construct pCACPNM1061 is identical to pCACPNM1097 except that the nucleotide sequence encoding the putative Metalloprotease is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated zinc Metalloprotease.
  • F. Immunization with pCACPNM908 [0261]
  • FIG. 36 and Table 10 show that mice immunized i.n. and i.m. with pCACPNM908 had chlamydial lung titers less than 40,000 in 3 of 6 cases at day 9 (mean 68,333) whereas the range of values for control mice sham immunized with saline was 56,000-424,100 IFU/lung (mean 207,962) at [0262] day 9. DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM569, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 215,600). The construct pCACPNM569 is identical to pCACPNM908 except that the nucleotide sequence encoding the putative CLP protease ATPase is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated signal peptidase.
  • G. Immunization with pCACPNM909 [0263]
  • FIG. 37 and Table 12 show that mice immunized i.n. and i.m. with pCACPNM909 had chlamydial lung titers less than 85,000 in 5 of 6 cases at day 9 (mean 87,683) whereas the range of values for control mice sham immunized with saline was 56,000-424,100 IFU/lung (mean 207,962) at [0264] day 9. DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM569, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 215,600). The construct pCACPNM569 is identical to pCACPNM909 except that the nucleotide sequence encoding the putative CLP protease subunit is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated signal peptidase.
  • H. Immunization with pCACPNM440 [0265]
  • FIG. 38 and Table 14 show that mice immunized i.n. and i.m. with pCACPNM440 had chlamydial lung titers less than 98,000 in 4 of 6 cases at day 9 (mean 87,616) whereas the range of values for control mice sham immunized with saline was 56,000-424,100 IFU/lung (mean 186,291) at [0266] day 9. DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM647 failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 143,883). The construct pCACPNM647 is identical to pCACPNM440 except that the nucleotide sequence encoding the putative transglycolase/transpeptidase gene is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated gene.
  • I. Immunization with pCACPNM459 [0267]
  • FIG. 39 and Table 16 show that mice immunized i.n. and i.m. with pCACPNM459 had chlamydial lung titers less than 70,000 in 4 of 6 cases at day 9 (mean 70,516) whereas the range of values for control mice sham immunized with saline was 56,000-424,100 IFU/lung (mean 186,291) at [0268] day 9. DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM647, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 143,883). The construct pCACPNM647 is identical to pCACPNM459 except that the nucleotide sequence encoding the putative CLPc protease is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated gene.
  • J. Immunization with pCACPNM708 [0269]
  • FIG. 40 and Table 18 show that mice immunized i.n. and i.m. with pCACPNM708 had chlamydial lung titers less than 52,000 in 4 of 6 cases at day 9 (mean 73,916) whereas the range of values for control mice sham immunized with saline was 56,000-424,100 IFU/lung (mean 207,962) at [0270] day 9. DNA immunisation per se was not responsible for the observed protective effect since another plasmid DNA construct, pCACPNM569, failed to protect, with lung titers in immunised mice similar to those obtained for saline-immunized control mice (mean 215,600). The construct pCACPNM569 is identical to pCACPNM708 except that the nucleotide sequence encoding the putative thioredoxin is replaced with a C. pneumoniae nucleotide sequence encoding an unrelated C. pneumoniae gene.
    • Example 4
  • This example illustrates the identification of B- and T-cell epitopes in proteins as expressed from each of pCACPNM213, pCACPNM882, pCACPNM208, pCACPNM1096, pCACPNM1097, pCACPNM909, pCACPNM440, pCACPNM459 and pCACPNM708. [0271]
  • B-cell epitopes were identified based on the product of flexibilty and hydrophobicity propensities using the program SEQSEE (Wishart D S, et al. “SEQSEE: a comprehensive program suite for protein sequence analysis.” [0272] Comput Appl Biosci. 1994 April;10(2):121-32) to identify external surface features (epitopes). T-cell epitopes for HLA-A0201 MHC subclass were identified based on the algorithm of Parker et al. 1995 (Parker K C, et al. “Peptide binding to MHC class I molecules: implications for antigenic peptide prediction.” Immunol Res 1995;14(1):34-57). These epitopes are shown in Tables 2, 5, 7, 9, 11, 13, 15, 17 and 19 and SEQ ID NOs: 41 to 74.
    TABLE 1
    BACTERIAL LOAD (INCLUSION
    FORMING UNITS PER LUNG) IN
    THE LUNGS OF BALB/C MICE
    IMMUNIZED WITH VARIOUS DNA
    IMMUNIZATION CONSTRUCTS
    IMMUNIZING CONSTRUCT
    Saline pCACPNM102 pCACPNM213
    MOUSE Day
    9 Day 9 Day 9
    1 64900 207500 54200
    2 116500 166500 10600
    3 34200 114700 67400
    4 377800 167400 32000
    5 86200 179700 66900
    6 206200 83900 79900
    7 142600
    8 103200
    MEAN 141450 153283.333 51833.3333
    SD 108598.7 45417.99 25908.35
    Wilcoxon p 1.655 0.0293
  • [0273]
    TABLE 2
    Identified B- T-cell epitopes from CPNM213
    B cell epitope T cell epitope
    188 VHHTLRESYKKGTPPST 434 WIAEYVSPV (SEQ ID No: 43)
    (SEQ ID No: 41)
    345 NLQKEISTEERQTKAR
    (SEQ ID No: 42)
  • [0274]
    TABLE 3
    BACTERIAL LOAD (INCLUSION
    FORMING UNITS PER LUNG) IN THE
    LUNGS OF BALB/C MICE IMMUNIZED
    WITH VARIOUS DNA IMMUNIZATION
    CONSTRUCTS
    IMMUNIZING CONSTRUCT
    Saline pCACPNM647 pCACPNM882
    MOUSE Day
    9 Day 9 Day 9
    1 209800 45100 18100
    2 70000 222000 130300
    3 226700 152500 72900
    4 178900 89000 53500
    5 424100 95500 63400
    6 242200 259200 126800
    7 256000
    8 56000
    9 173600
    10  185000
    11  121400
    12  91800
    MEAN 186291.667 143883.333 77500
    SD 100263.3 83169.31 43686.75
    Wilcoxon p 0.4936 0.0182
  • [0275]
    TABLE 4
    BACTERIAL LOAD (INCLUSION
    FORMING UNITS PER LUNG) IN THE
    LUNGS OF BALB/C MICE IMMUNIZED
    WITH VARIOUS DNA IMMUNIZATION
    CONSTRUCTS
    IMMUNIZING CONSTRUCT
    Saline pCACPNM647 pCACPNM208
    MOUSE Day
    9 Day 9 Day 9
    1 209800 45100 142500
    2 70000 222000 66900
    3 226700 152500 58200
    4 178900 89000 46500
    5 424100 95500 110900
    6 242200 259200 65600
    7 256000
    8 56000
    9 173600
    10  185000
    11  121400
    12  91800
    MEAN 186291.667 143883.333 81766.6667
    SD 100263.3 83169.31 36929.10
    Wilcoxon p 0.4936 0.0135
  • [0276]
    TABLE 5
    Identified B- T-cell epitopes from CPNM208
    B cell epitope T cell epitope
    220 KGNNSSPRSPAP 67 LLIEDMDLI
    (SEQ ID No: 44) (SEQ ID No: 46)
    313 GENFQKNSS 66 NLLIEDMDL
    (SEQ ID No: 45) (SEQ ID No: 47)
  • [0277]
    TABLE 6
    BACTERIAL LOAD (INCLUSION
    FORMING UNITS PER LUNG) IN THE
    LUNGS OF BALB/C MICE IMMUNIZED
    WITH VARIOUS DNA IMMUNIZATION
    CONSTRUCTS
    IMMUNIZING CONSTRUCT
    Saline pCACPNM553 pCACPNM1096
    MOUSE Day
    9 Day 9 Day 9
    1 136900 135600 21000
    2 81700 112600 9700
    3 119400 88600 28500
    4 58500 121700 52000
    5 110600 165300 17200
    6 51300 45700 21600
    7 170000
    8 112800
    MEAN 105150 111583.333 25000
    SD 39876.3 41071.91 14585.88
    Wilcoxon p 1.245 0.0013
  • [0278]
    TABLE 7
    Identified B- T-cell epitopes from CPNM1096
    B cell epitope T cell epitope
    328 TDLEGLEEDHKDSPWE 135 YLGDEILEV
    (SEQ ID No: 48) (SEQ ID No: 50)
    589 SENAKKSEEQTSPQETPE 373 YLYSLLSML
    (SEQ ID No: 49) (SEQ ID No: 51)
  • [0279]
    TABLE 8
    BACTERIAL LOAD (INCLUSION FORMING
    UNITS PER LUNG) IN THE LUNGS OF
    BALB/C MICE IMMUNIZED WITH
    VARIOUS DNA IMMUNIZATION
    CONSTRUCTS
    IMMUNIZING CONSTRUCT
    Saline pCACPNM1061 pCACPNM1097
    MOUSE Day
    9 Day 9 Day 9
    1 232900 120800 50300
    2 168100 184100 43900
    3 105500 95600 65200
    4 173100 147500 157900
    5 90000 218700 22800
    6 242100 124700 37200
    7 183700
    8 134900
    MEAN 166287.5 148566.667 62883.3333
    SD 54821.4 45450.00 48618.00
    Wilcoxon p 0.662 0.0047
  • [0280]
    TABLE 9
    Identified B- T-cell epitopes from CPNM1097
    B cell epitope T cell epitope
    198 TTNRQKAL 207 SVLSRVNYV
    (SEQ ID No: 52) (SEQ ID No: 54)
    221 VNSSNSNRLRE 279 KLSSLIPGL
    (SEQ ID No: 53) (SEQ ID No: 55)
    118 ILIGHKKHV
    (SEQ ID No: 56)
  • [0281]
    TABLE 10
    BACTERIAL LOAD (INCLUSION
    FORMING UNITS PER LUNG)
    IN THE LUNGS OF BALB/C MICE
    IMMUNIZED WITH VARIOUS DNA
    IMMUNIZATION CONSTRUCTS
    IMMUNIZING CONSTRUCT
    Saline pCACPNM569 pCACPNM908
    MOUSE Day
    9 Day 9 Day 9
    1 209800 142800 37300
    2 70000 420700 85000
    3 226700 116600 35700
    4 178900 161300 39700
    5 424100 89200 123400
    6 242200 363000 88900
    7 256000
    8 56000
    MEAN 207962.5 215600 68333.3333
    SD 115585.8 139870.70 36279.40
    Wilcoxon p 0.8518 0.02
  • [0282]
    TABLE 11
    Identified B- T-cell epitopes from CPNM908
    B cell epitope T cell epitope
    226 PPKGGRKHPNQEYI 137 KILDVPFTI
    (SEQ ID No: 57) (SEQ ID No: 59)
    273 SDDQADLSQKTRDH 168 LLQAADYDV
    (SEQ ID No: 58) (SEQ ID No: 60)
  • [0283]
    TABLE 12
    BACTERIAL LOAD (INCLUSION
    FORMING UNITS PER LUNG) IN THE
    LUNGS OF BALB/C MICE IMMUNIZED
    WITH VARIOUS DNA IMMUNIZATION
    CONSTRUCTS
    IMMUNIZING CONSTRUCT
    Saline pCACPNM569 PCACPNM909
    MOUSE Day
    9 Day 9 Day 9
    1 209800 142800 206700
    2 70000 420700 84700
    3 226700 116600 81100
    4 178900 161300 56700
    5 424100 89200 53900
    6 242200 363000 43000
    7 256000
    8 56000
    MEAN 207962.5 215600 87683.3333
    SD 115585.8 139870.70 60522.87
    Wilcoxon p 0.8518 0.0426
  • [0284]
    TABLE 13
    Identified B- T-cell epitopes from CPNM909
    B cell epitope T cell epitope
    107 GTKGKRHAL 76 AIYDTIRFL
    (SEQ ID No: 61) (SEQ ID No: 63)
    193 AKETNKDTSST
    (SEQ ID No: 62)
  • [0285]
    TABLE 14
    BACTERIAL LOAD (INCLUSION
    FORMING UNITS PER LUNG) IN THE
    LUNGS OF BALB/C MICE IMMUNIZED
    WITH VARIOUS DNA IMMUNIZATION
    CONSTRUCTS
    IMMUNIZING CONSTRUCT
    Saline pCACPNM647 pCACPNM440
    MOUSE Day
    9 Day 9 Day 9
    1 209800 45100 97200
    2 70000 222000 92500
    3 226700 152500 104400
    4 178900 89000 60900
    5 424100 95500 40400
    6 242200 259200 130300
    7 256000
    8 56000
    9 173600
    10  185000
    11  121400
    12  91800
    MEAN 186291.667 143883.333 87616.6667
    SD 100263.3 83169.31 32132.31
    Wilcoxon p 0.4936 0.0415
  • [0286]
    TABLE 15
    Identified B- T-cell epitopes from CPNM440
    B cell epitope T cell epitope
    287 DPTNYKEYFNNKERIEHTK  40 ALGQHEFCV
    (SEQ ID No: 64) (SEQ ID No: 66)
    637 KRLYEEWNRSPKQGGTR 456 ILATGIQMV
    (SEQ ID No: 65) (SEQ ID No: 67)
  • [0287]
    TABLE 16
    BACTERIAL LOAD (INCLUSION
    FORMING UNITS PER LUNG) IN THE
    LUNGS OF BALB/C MICE IMMUNIZED
    WITH VARIOUS DNA IMMUNIZATION
    CONSTRUCTS
    IMMUNIZING CONSTRUCT
    Saline pCACPNM647 pCACPNTA459
    MOUSE Day
    9 Day 9 Day 9
    1 209800 45100 77400
    2 70000 222000 60700
    3 226700 152500 121000
    4 178900 89000 68500
    5 424100 95500 44800
    6 242200 259200 50700
    7 256000
    8 56000
    9 173600
    10  185000
    11  121400
    12  91800
    MEAN 186291.667 143883.333 70516.6667
    SD 100263.3 83169.31 27387.69
    Wilcoxon p 0.4936 0.0047
  • [0288]
    TABLE 17
    Identified B- T-cell epitopes from CPNM459
    B cell epitope T cell epitope
    467 DEEKKLRERLQSMKQEWENHKEEHQ 565 FLFLGPTGV
    (SEQ ID No: 68) (SEQ ID No: 70)
    548 IRRSRTGIKDPNRPTG 410 FLPDKAIDL
    (SEQ ID No: 69) (SEQ ID No: 71)
  • [0289]
    TABLE 18
    BACTERIAL LOAD (INCLUSION
    FORMING UNITS PER LUNG) IN THE
    LUNGS OF BALB/C MICE IMMUNIZED
    WITH VARIOUS DNA IMMUNIZATION
    CONSTRUCTS
    IMMUNIZING CONSTRUCT
    Saline pCACPNM569 pCACPNM708
    MOUSE Day
    9 Day 9 Day 9
    1 209800 142800 95100
    2 70000 420700 189600
    3 226700 116600 29000
    4 178900 161300 51400
    5 424100 89200 31500
    6 242200 363000 46900
    7 256000
    8 56000
    MEAN 207962.5 215600 73916.6667
    SD 115585.8 139870.70 61457.22
    Wilcoxon p 0.8518 0.0127
  • [0290]
    TABLE 19
    Identified B- T-cell epitopes from pCPNM708
    B cell epitope T cell epitope
    54 NIDENSKPAETYE 40 NLAAELPHV
    (SEQ ID No: 72) (SEQ ID No: 73)
    74 ILFKDGNEV
    (SEQ ID No: 74)
  • [0291]
  • 1 74 1 1787 DNA Chlamydia pneumoniae CDS (101)..(1684) 1 aatctcattc ccccatcgac taaatccacc acggactccg acctcccatg tcttcaatcc 60 atatgaacgt aatattaagt agcaaattga gtactatata atg aag atg cat agg 115 Met Lys Met His Arg 1 5 ctt aaa cct acc tta aaa agt ctg atc cct aat ctt ctt ttc tta ttg 163 Leu Lys Pro Thr Leu Lys Ser Leu Ile Pro Asn Leu Leu Phe Leu Leu 10 15 20 ctc act ctt tca agc tgc tca aag caa aaa caa gaa ccc tta gga aaa 211 Leu Thr Leu Ser Ser Cys Ser Lys Gln Lys Gln Glu Pro Leu Gly Lys 25 30 35 cat ctc gtt att gcg atg agc cat gat ctc gcc gac cta gat cct cgc 259 His Leu Val Ile Ala Met Ser His Asp Leu Ala Asp Leu Asp Pro Arg 40 45 50 aat gcc tat tta agc aga gat gct tcc cta gca aaa gcc ctc tat gaa 307 Asn Ala Tyr Leu Ser Arg Asp Ala Ser Leu Ala Lys Ala Leu Tyr Glu 55 60 65 gga ctg aca aga gaa act gat caa gga atc gca ctg gct ctt gca gaa 355 Gly Leu Thr Arg Glu Thr Asp Gln Gly Ile Ala Leu Ala Leu Ala Glu 70 75 80 85 agt tat acc ctg tca aaa gat cat aag gtc tat acc ttt aaa ctc aga 403 Ser Tyr Thr Leu Ser Lys Asp His Lys Val Tyr Thr Phe Lys Leu Arg 90 95 100 cct tct gtg tgg agc gat ggc act cca ctc act gct tat gac ttt gaa 451 Pro Ser Val Trp Ser Asp Gly Thr Pro Leu Thr Ala Tyr Asp Phe Glu 105 110 115 aaa tct ata aaa caa ctg tac ttc gaa gaa ttt tca cct tcc ata cat 499 Lys Ser Ile Lys Gln Leu Tyr Phe Glu Glu Phe Ser Pro Ser Ile His 120 125 130 act tta ctc ggc gtg att aaa aat tct tcg gca atc cac aat gct caa 547 Thr Leu Leu Gly Val Ile Lys Asn Ser Ser Ala Ile His Asn Ala Gln 135 140 145 aaa tct ctg gaa act ctt ggg ata cag gca aaa gat gat ctt act ttg 595 Lys Ser Leu Glu Thr Leu Gly Ile Gln Ala Lys Asp Asp Leu Thr Leu 150 155 160 165 gtg att acc cta gag caa cct ttc cca tac ttt ctc aca ctt atc gct 643 Val Ile Thr Leu Glu Gln Pro Phe Pro Tyr Phe Leu Thr Leu Ile Ala 170 175 180 cgc ccc gta ttc tcc cct gtt cat cac acc ctt agg gaa tcc tat aag 691 Arg Pro Val Phe Ser Pro Val His His Thr Leu Arg Glu Ser Tyr Lys 185 190 195 aaa gga aca ccc cca tcc aca tac atc tcc aat ggg ccc ttt gtc tta 739 Lys Gly Thr Pro Pro Ser Thr Tyr Ile Ser Asn Gly Pro Phe Val Leu 200 205 210 aaa aaa cat gaa cac caa aac tac tta att tta gaa aaa aat cct cac 787 Lys Lys His Glu His Gln Asn Tyr Leu Ile Leu Glu Lys Asn Pro His 215 220 225 tac tat gat cat gaa tca gta aag tta gac cga gtc acc tta aaa att 835 Tyr Tyr Asp His Glu Ser Val Lys Leu Asp Arg Val Thr Leu Lys Ile 230 235 240 245 atc cca gac gcc tcc aca gcc acg aaa ctt ttc aaa agt aaa tct ata 883 Ile Pro Asp Ala Ser Thr Ala Thr Lys Leu Phe Lys Ser Lys Ser Ile 250 255 260 gat tgg att ggc tca cct tgg agc gct ccg ata tct aac gaa gac caa 931 Asp Trp Ile Gly Ser Pro Trp Ser Ala Pro Ile Ser Asn Glu Asp Gln 265 270 275 aaa gtt ctc tcc caa gaa aag att ctt acc tat tct gtt tca agc acc 979 Lys Val Leu Ser Gln Glu Lys Ile Leu Thr Tyr Ser Val Ser Ser Thr 280 285 290 acc ctt ctt atc tat aac ctg caa aaa cct cta ata caa aat aaa gcc 1027 Thr Leu Leu Ile Tyr Asn Leu Gln Lys Pro Leu Ile Gln Asn Lys Ala 295 300 305 ctc agg aaa gcc att gct cat gct att gat aga aaa tct atc tta aga 1075 Leu Arg Lys Ala Ile Ala His Ala Ile Asp Arg Lys Ser Ile Leu Arg 310 315 320 325 ctc gtg cct tca gga caa gaa gct gta act cta gtt ccc cca aat ctt 1123 Leu Val Pro Ser Gly Gln Glu Ala Val Thr Leu Val Pro Pro Asn Leu 330 335 340 tca caa ctc aat ctt caa aaa gag atc tca aca gaa gaa cga caa aca 1171 Ser Gln Leu Asn Leu Gln Lys Glu Ile Ser Thr Glu Glu Arg Gln Thr 345 350 355 aaa gcc aga gca tat ttt caa gaa gct aaa gaa aca ctt tct gaa aaa 1219 Lys Ala Arg Ala Tyr Phe Gln Glu Ala Lys Glu Thr Leu Ser Glu Lys 360 365 370 gaa ctc gca gaa ctc agc atc ctc tat cct ata gat tcc tcg aat tcc 1267 Glu Leu Ala Glu Leu Ser Ile Leu Tyr Pro Ile Asp Ser Ser Asn Ser 375 380 385 tcc atc ata gct caa gaa atc caa aga caa ctt aaa gat acc tta gga 1315 Ser Ile Ile Ala Gln Glu Ile Gln Arg Gln Leu Lys Asp Thr Leu Gly 390 395 400 405 ttg aaa atc aaa atc caa ggc atg gag tac cac tgc ttt tta aag aaa 1363 Leu Lys Ile Lys Ile Gln Gly Met Glu Tyr His Cys Phe Leu Lys Lys 410 415 420 cgt cgt caa gga gat ttc ttc ata gcg aca gga gga tgg att gcg gaa 1411 Arg Arg Gln Gly Asp Phe Phe Ile Ala Thr Gly Gly Trp Ile Ala Glu 425 430 435 tac gta agc ccc gta gcc ttc cta tct att cta ggc aac ccc aga gac 1459 Tyr Val Ser Pro Val Ala Phe Leu Ser Ile Leu Gly Asn Pro Arg Asp 440 445 450 ctc aca caa tgg aga aac agt gat tac gaa aag act tta gag aaa ctc 1507 Leu Thr Gln Trp Arg Asn Ser Asp Tyr Glu Lys Thr Leu Glu Lys Leu 455 460 465 tat ctc cct cat gcc tac aaa gag aat tta aaa cgc gca gaa atg ata 1555 Tyr Leu Pro His Ala Tyr Lys Glu Asn Leu Lys Arg Ala Glu Met Ile 470 475 480 485 ata gaa gaa gaa acc ccg att atc ccc ctg tat cac ggc aaa tat att 1603 Ile Glu Glu Glu Thr Pro Ile Ile Pro Leu Tyr His Gly Lys Tyr Ile 490 495 500 tac gct ata cat cct aaa atc cag aat aca ttc gga tct ctt cta ggc 1651 Tyr Ala Ile His Pro Lys Ile Gln Asn Thr Phe Gly Ser Leu Leu Gly 505 510 515 cac aca gat ctc aaa aat atc gat atc tta agt tagatccgaa atggaaaaat 1704 His Thr Asp Leu Lys Asn Ile Asp Ile Leu Ser 520 525 taaaaatttt atagacaatc ttgaaaagag aattaaaaat ttttaattta aattatagtt 1764 gcaattgaaa acgcccctaa gaa 1787 2 528 PRT Chlamydia pneumoniae SITE (188)...(61) B-cell epitope 2 Met Lys Met His Arg Leu Lys Pro Thr Leu Lys Ser Leu Ile Pro Asn 1 5 10 15 Leu Leu Phe Leu Leu Leu Thr Leu Ser Ser Cys Ser Lys Gln Lys Gln 20 25 30 Glu Pro Leu Gly Lys His Leu Val Ile Ala Met Ser His Asp Leu Ala 35 40 45 Asp Leu Asp Pro Arg Asn Ala Tyr Leu Ser Arg Asp Ala Ser Leu Ala 50 55 60 Lys Ala Leu Tyr Glu Gly Leu Thr Arg Glu Thr Asp Gln Gly Ile Ala 65 70 75 80 Leu Ala Leu Ala Glu Ser Tyr Thr Leu Ser Lys Asp His Lys Val Tyr 85 90 95 Thr Phe Lys Leu Arg Pro Ser Val Trp Ser Asp Gly Thr Pro Leu Thr 100 105 110 Ala Tyr Asp Phe Glu Lys Ser Ile Lys Gln Leu Tyr Phe Glu Glu Phe 115 120 125 Ser Pro Ser Ile His Thr Leu Leu Gly Val Ile Lys Asn Ser Ser Ala 130 135 140 Ile His Asn Ala Gln Lys Ser Leu Glu Thr Leu Gly Ile Gln Ala Lys 145 150 155 160 Asp Asp Leu Thr Leu Val Ile Thr Leu Glu Gln Pro Phe Pro Tyr Phe 165 170 175 Leu Thr Leu Ile Ala Arg Pro Val Phe Ser Pro Val His His Thr Leu 180 185 190 Arg Glu Ser Tyr Lys Lys Gly Thr Pro Pro Ser Thr Tyr Ile Ser Asn 195 200 205 Gly Pro Phe Val Leu Lys Lys His Glu His Gln Asn Tyr Leu Ile Leu 210 215 220 Glu Lys Asn Pro His Tyr Tyr Asp His Glu Ser Val Lys Leu Asp Arg 225 230 235 240 Val Thr Leu Lys Ile Ile Pro Asp Ala Ser Thr Ala Thr Lys Leu Phe 245 250 255 Lys Ser Lys Ser Ile Asp Trp Ile Gly Ser Pro Trp Ser Ala Pro Ile 260 265 270 Ser Asn Glu Asp Gln Lys Val Leu Ser Gln Glu Lys Ile Leu Thr Tyr 275 280 285 Ser Val Ser Ser Thr Thr Leu Leu Ile Tyr Asn Leu Gln Lys Pro Leu 290 295 300 Ile Gln Asn Lys Ala Leu Arg Lys Ala Ile Ala His Ala Ile Asp Arg 305 310 315 320 Lys Ser Ile Leu Arg Leu Val Pro Ser Gly Gln Glu Ala Val Thr Leu 325 330 335 Val Pro Pro Asn Leu Ser Gln Leu Asn Leu Gln Lys Glu Ile Ser Thr 340 345 350 Glu Glu Arg Gln Thr Lys Ala Arg Ala Tyr Phe Gln Glu Ala Lys Glu 355 360 365 Thr Leu Ser Glu Lys Glu Leu Ala Glu Leu Ser Ile Leu Tyr Pro Ile 370 375 380 Asp Ser Ser Asn Ser Ser Ile Ile Ala Gln Glu Ile Gln Arg Gln Leu 385 390 395 400 Lys Asp Thr Leu Gly Leu Lys Ile Lys Ile Gln Gly Met Glu Tyr His 405 410 415 Cys Phe Leu Lys Lys Arg Arg Gln Gly Asp Phe Phe Ile Ala Thr Gly 420 425 430 Gly Trp Ile Ala Glu Tyr Val Ser Pro Val Ala Phe Leu Ser Ile Leu 435 440 445 Gly Asn Pro Arg Asp Leu Thr Gln Trp Arg Asn Ser Asp Tyr Glu Lys 450 455 460 Thr Leu Glu Lys Leu Tyr Leu Pro His Ala Tyr Lys Glu Asn Leu Lys 465 470 475 480 Arg Ala Glu Met Ile Ile Glu Glu Glu Thr Pro Ile Ile Pro Leu Tyr 485 490 495 His Gly Lys Tyr Ile Tyr Ala Ile His Pro Lys Ile Gln Asn Thr Phe 500 505 510 Gly Ser Leu Leu Gly His Thr Asp Leu Lys Asn Ile Asp Ile Leu Ser 515 520 525 3 1226 DNA Chlamydia pneumoniae CDS (101)..(1123) 3 ttccagagaa atcctgatcc tgaaaaactt cctgaaacaa ttgctttaac tataacacgg 60 gaacctaaag catatcctcc aaggacgtta acataccaat ttg cgg ttg gga aat 115 Leu Arg Leu Gly Asn 1 5 aag cct atg caa cct ttt atc ttt act tta ctg tgc ttg aca tct ttg 163 Lys Pro Met Gln Pro Phe Ile Phe Thr Leu Leu Cys Leu Thr Ser Leu 10 15 20 gtt tct tta gtc gcc ttt gat gct gcg aat gct cgt aaa cgt tgt gcc 211 Val Ser Leu Val Ala Phe Asp Ala Ala Asn Ala Arg Lys Arg Cys Ala 25 30 35 tgt gct caa act ata gaa cgt gga gag aac ttc ttt tcc ata aaa cgc 259 Cys Ala Gln Thr Ile Glu Arg Gly Glu Asn Phe Phe Ser Ile Lys Arg 40 45 50 tct gct tgt gct gaa atc gaa tat caa gaa aaa tct cgc cac gcc tca 307 Ser Ala Cys Ala Glu Ile Glu Tyr Gln Glu Lys Ser Arg His Ala Ser 55 60 65 gca att gaa aga atc tca aaa gat aaa ggc aaa gtc act cca aag cag 355 Ala Ile Glu Arg Ile Ser Lys Asp Lys Gly Lys Val Thr Pro Lys Gln 70 75 80 85 att gcg aaa gta gct act aag aaa aag caa aga tac cgt tta ttg cag 403 Ile Ala Lys Val Ala Thr Lys Lys Lys Gln Arg Tyr Arg Leu Leu Gln 90 95 100 gtt cct ttt tca agg cct ccg aat aac tca agg tat aac ctc tat gct 451 Val Pro Phe Ser Arg Pro Pro Asn Asn Ser Arg Tyr Asn Leu Tyr Ala 105 110 115 ttg ctt agt gaa cct ccc gaa tgc tat agc gat aca gca tca tgg tat 499 Leu Leu Ser Glu Pro Pro Glu Cys Tyr Ser Asp Thr Ala Ser Trp Tyr 120 125 130 gct att ttt att cgg tta ctt cga cgt gct tat gta gac acg gga aat 547 Ala Ile Phe Ile Arg Leu Leu Arg Arg Ala Tyr Val Asp Thr Gly Asn 135 140 145 gta cct cct gga tct gag tat gcc atc gct aat gct ttg ata agt aac 595 Val Pro Pro Gly Ser Glu Tyr Ala Ile Ala Asn Ala Leu Ile Ser Asn 150 155 160 165 aaa caa gag att tta gag agg gga gcg cag ctt gga ccc gat gtt att 643 Lys Gln Glu Ile Leu Glu Arg Gly Ala Gln Leu Gly Pro Asp Val Ile 170 175 180 gaa act cta aca ttg cct gag gaa caa gcc gag att ttt tat aaa atg 691 Glu Thr Leu Thr Leu Pro Glu Glu Gln Ala Glu Ile Phe Tyr Lys Met 185 190 195 ctc aaa ggg tcg tca aac tct cag tcg cta ctg aat ttt ctg cat tat 739 Leu Lys Gly Ser Ser Asn Ser Gln Ser Leu Leu Asn Phe Leu His Tyr 200 205 210 gaa gag aaa agc tta ggc cac tgt aag cta aat ctg atc ttc atg gat 787 Glu Glu Lys Ser Leu Gly His Cys Lys Leu Asn Leu Ile Phe Met Asp 215 220 225 ccc cta ctg tta gaa gct gtt cta gat cat ccc gat gct tat agg gaa 835 Pro Leu Leu Leu Glu Ala Val Leu Asp His Pro Asp Ala Tyr Arg Glu 230 235 240 245 acg tcg ctc ctg cgc gat ggc att tgg gaa gcg gtg aag cgt caa gaa 883 Thr Ser Leu Leu Arg Asp Gly Ile Trp Glu Ala Val Lys Arg Gln Glu 250 255 260 cat gcc atc caa gaa cat ggc cag gca gct gct ttg gag ctt ttt aaa 931 His Ala Ile Gln Glu His Gly Gln Ala Ala Ala Leu Glu Leu Phe Lys 265 270 275 aca cgc acc gac ttc cgc ctg gag ctg cga gat aag atg cag tta ctt 979 Thr Arg Thr Asp Phe Arg Leu Glu Leu Arg Asp Lys Met Gln Leu Leu 280 285 290 cta agt cga tac gat ttg ctc ccc tta tta aat aaa aaa atg ttc gac 1027 Leu Ser Arg Tyr Asp Leu Leu Pro Leu Leu Asn Lys Lys Met Phe Asp 295 300 305 tac acc tta gga agt gcc gga gat tac tta ttt ttg gta gac cca gat 1075 Tyr Thr Leu Gly Ser Ala Gly Asp Tyr Leu Phe Leu Val Asp Pro Asp 310 315 320 325 act aag gca att tct cga tgt cgc tgc cct tca aag agt att aaa tta 1123 Thr Lys Ala Ile Ser Arg Cys Arg Cys Pro Ser Lys Ser Ile Lys Leu 330 335 340 taatttaatt ttaatattta ttttaaatag ttttttttga taattgtctt aataagtact 1183 ataaaaaata tttctatagg taggaccatg gcagacgaga ccc 1226 4 341 PRT Chlamydia pneumoniae 4 Leu Arg Leu Gly Asn Lys Pro Met Gln Pro Phe Ile Phe Thr Leu Leu 1 5 10 15 Cys Leu Thr Ser Leu Val Ser Leu Val Ala Phe Asp Ala Ala Asn Ala 20 25 30 Arg Lys Arg Cys Ala Cys Ala Gln Thr Ile Glu Arg Gly Glu Asn Phe 35 40 45 Phe Ser Ile Lys Arg Ser Ala Cys Ala Glu Ile Glu Tyr Gln Glu Lys 50 55 60 Ser Arg His Ala Ser Ala Ile Glu Arg Ile Ser Lys Asp Lys Gly Lys 65 70 75 80 Val Thr Pro Lys Gln Ile Ala Lys Val Ala Thr Lys Lys Lys Gln Arg 85 90 95 Tyr Arg Leu Leu Gln Val Pro Phe Ser Arg Pro Pro Asn Asn Ser Arg 100 105 110 Tyr Asn Leu Tyr Ala Leu Leu Ser Glu Pro Pro Glu Cys Tyr Ser Asp 115 120 125 Thr Ala Ser Trp Tyr Ala Ile Phe Ile Arg Leu Leu Arg Arg Ala Tyr 130 135 140 Val Asp Thr Gly Asn Val Pro Pro Gly Ser Glu Tyr Ala Ile Ala Asn 145 150 155 160 Ala Leu Ile Ser Asn Lys Gln Glu Ile Leu Glu Arg Gly Ala Gln Leu 165 170 175 Gly Pro Asp Val Ile Glu Thr Leu Thr Leu Pro Glu Glu Gln Ala Glu 180 185 190 Ile Phe Tyr Lys Met Leu Lys Gly Ser Ser Asn Ser Gln Ser Leu Leu 195 200 205 Asn Phe Leu His Tyr Glu Glu Lys Ser Leu Gly His Cys Lys Leu Asn 210 215 220 Leu Ile Phe Met Asp Pro Leu Leu Leu Glu Ala Val Leu Asp His Pro 225 230 235 240 Asp Ala Tyr Arg Glu Thr Ser Leu Leu Arg Asp Gly Ile Trp Glu Ala 245 250 255 Val Lys Arg Gln Glu His Ala Ile Gln Glu His Gly Gln Ala Ala Ala 260 265 270 Leu Glu Leu Phe Lys Thr Arg Thr Asp Phe Arg Leu Glu Leu Arg Asp 275 280 285 Lys Met Gln Leu Leu Leu Ser Arg Tyr Asp Leu Leu Pro Leu Leu Asn 290 295 300 Lys Lys Met Phe Asp Tyr Thr Leu Gly Ser Ala Gly Asp Tyr Leu Phe 305 310 315 320 Leu Val Asp Pro Asp Thr Lys Ala Ile Ser Arg Cys Arg Cys Pro Ser 325 330 335 Lys Ser Ile Lys Leu 340 5 1235 DNA Chlamydia pneumoniae CDS (101)..(1132) 5 gttacttttt ttttcataaa aaccccatgt aacttttact tgctcatatt gagaagtccc 60 ccatactata aaaggcaacg ttttcttttc ttggtttttt atg ctc acc cta ggc 115 Met Leu Thr Leu Gly 1 5 ttg gaa agt tct tgc gat gag act gcc tgc gct ata gtt aat gag gat 163 Leu Glu Ser Ser Cys Asp Glu Thr Ala Cys Ala Ile Val Asn Glu Asp 10 15 20 aag cag ata tta gca aat att att gcc tct caa gat atc cat gca tcc 211 Lys Gln Ile Leu Ala Asn Ile Ile Ala Ser Gln Asp Ile His Ala Ser 25 30 35 tat ggc gga gtc gtt cct gaa ctt gct tca aga gca cat ctc cat atc 259 Tyr Gly Gly Val Val Pro Glu Leu Ala Ser Arg Ala His Leu His Ile 40 45 50 ttc cca caa gtg ata aat aaa gct cta caa cag gcc aac tta ttg atc 307 Phe Pro Gln Val Ile Asn Lys Ala Leu Gln Gln Ala Asn Leu Leu Ile 55 60 65 gaa gat atg gat ctg att gca gta acg caa act cca ggg ttg ata ggt 355 Glu Asp Met Asp Leu Ile Ala Val Thr Gln Thr Pro Gly Leu Ile Gly 70 75 80 85 tct cta tca gta gga gtg cat ttt ggt aaa ggc att gcc ata gga gca 403 Ser Leu Ser Val Gly Val His Phe Gly Lys Gly Ile Ala Ile Gly Ala 90 95 100 aaa aaa tcc ttg att gga gtc aat cac gtc gaa gct cat ctc tat gct 451 Lys Lys Ser Leu Ile Gly Val Asn His Val Glu Ala His Leu Tyr Ala 105 110 115 gcc tat atg gca gcg caa aac gtg caa ttc cct gct tta ggt ctt gtg 499 Ala Tyr Met Ala Ala Gln Asn Val Gln Phe Pro Ala Leu Gly Leu Val 120 125 130 gtc tct gga gct cat acc gca gcg ttt ttt ata gaa aat cct aca tcc 547 Val Ser Gly Ala His Thr Ala Ala Phe Phe Ile Glu Asn Pro Thr Ser 135 140 145 tat aaa ctc ata gga aaa act cga gat gat gct ata gga gaa act ttt 595 Tyr Lys Leu Ile Gly Lys Thr Arg Asp Asp Ala Ile Gly Glu Thr Phe 150 155 160 165 gat aaa gta gga cgc ttt cta gga tta cca tac cct gca ggc cca tta 643 Asp Lys Val Gly Arg Phe Leu Gly Leu Pro Tyr Pro Ala Gly Pro Leu 170 175 180 att gaa aaa ctc gct tta gaa ggc tct gag gac agt tat cct ttt agt 691 Ile Glu Lys Leu Ala Leu Glu Gly Ser Glu Asp Ser Tyr Pro Phe Ser 185 190 195 cca gct aaa gtc cca aac tat gac ttt tca ttc agc ggt ctt aaa aca 739 Pro Ala Lys Val Pro Asn Tyr Asp Phe Ser Phe Ser Gly Leu Lys Thr 200 205 210 gct gtt ctc tac gca atc aaa gga aat aat agt agc ccc cgc tct cct 787 Ala Val Leu Tyr Ala Ile Lys Gly Asn Asn Ser Ser Pro Arg Ser Pro 215 220 225 gct cca gag ata tct tta gaa aaa caa aga gat atc gct gct tca ttt 835 Ala Pro Glu Ile Ser Leu Glu Lys Gln Arg Asp Ile Ala Ala Ser Phe 230 235 240 245 caa aaa gcg gcc tgc act act att gca caa aaa ctt ccc act att ata 883 Gln Lys Ala Ala Cys Thr Thr Ile Ala Gln Lys Leu Pro Thr Ile Ile 250 255 260 aaa gaa ttt tcg tgc cga tct ata ctt att gga ggt ggc gta gcc att 931 Lys Glu Phe Ser Cys Arg Ser Ile Leu Ile Gly Gly Gly Val Ala Ile 265 270 275 aat gaa tac ttt aga tcc gca ata caa act gcg tgt aat cta cct gta 979 Asn Glu Tyr Phe Arg Ser Ala Ile Gln Thr Ala Cys Asn Leu Pro Val 280 285 290 tac ttc ccc cct gct aaa cta tgc tca gat aat gct gct atg att gca 1027 Tyr Phe Pro Pro Ala Lys Leu Cys Ser Asp Asn Ala Ala Met Ile Ala 295 300 305 ggt cta ggg gga gaa aat ttt caa aaa aac tct agt att ccg gaa att 1075 Gly Leu Gly Gly Glu Asn Phe Gln Lys Asn Ser Ser Ile Pro Glu Ile 310 315 320 325 cgt ata tgc gca aga tat cag tgg gaa tct gta tca cca ttc tcc tta 1123 Arg Ile Cys Ala Arg Tyr Gln Trp Glu Ser Val Ser Pro Phe Ser Leu 330 335 340 gcc tct ccg tagtcctcca aggctgcaag gagtccagtc actcctctac 1172 Ala Ser Pro atctcgggga gaactcgcta ttaatataag agatgaaccc cgttctttag atccaagaca 1232 agt 1235 6 344 PRT Chlamydia pneumoniae SITE (220)...(231) B-cell epitope 6 Met Leu Thr Leu Gly Leu Glu Ser Ser Cys Asp Glu Thr Ala Cys Ala 1 5 10 15 Ile Val Asn Glu Asp Lys Gln Ile Leu Ala Asn Ile Ile Ala Ser Gln 20 25 30 Asp Ile His Ala Ser Tyr Gly Gly Val Val Pro Glu Leu Ala Ser Arg 35 40 45 Ala His Leu His Ile Phe Pro Gln Val Ile Asn Lys Ala Leu Gln Gln 50 55 60 Ala Asn Leu Leu Ile Glu Asp Met Asp Leu Ile Ala Val Thr Gln Thr 65 70 75 80 Pro Gly Leu Ile Gly Ser Leu Ser Val Gly Val His Phe Gly Lys Gly 85 90 95 Ile Ala Ile Gly Ala Lys Lys Ser Leu Ile Gly Val Asn His Val Glu 100 105 110 Ala His Leu Tyr Ala Ala Tyr Met Ala Ala Gln Asn Val Gln Phe Pro 115 120 125 Ala Leu Gly Leu Val Val Ser Gly Ala His Thr Ala Ala Phe Phe Ile 130 135 140 Glu Asn Pro Thr Ser Tyr Lys Leu Ile Gly Lys Thr Arg Asp Asp Ala 145 150 155 160 Ile Gly Glu Thr Phe Asp Lys Val Gly Arg Phe Leu Gly Leu Pro Tyr 165 170 175 Pro Ala Gly Pro Leu Ile Glu Lys Leu Ala Leu Glu Gly Ser Glu Asp 180 185 190 Ser Tyr Pro Phe Ser Pro Ala Lys Val Pro Asn Tyr Asp Phe Ser Phe 195 200 205 Ser Gly Leu Lys Thr Ala Val Leu Tyr Ala Ile Lys Gly Asn Asn Ser 210 215 220 Ser Pro Arg Ser Pro Ala Pro Glu Ile Ser Leu Glu Lys Gln Arg Asp 225 230 235 240 Ile Ala Ala Ser Phe Gln Lys Ala Ala Cys Thr Thr Ile Ala Gln Lys 245 250 255 Leu Pro Thr Ile Ile Lys Glu Phe Ser Cys Arg Ser Ile Leu Ile Gly 260 265 270 Gly Gly Val Ala Ile Asn Glu Tyr Phe Arg Ser Ala Ile Gln Thr Ala 275 280 285 Cys Asn Leu Pro Val Tyr Phe Pro Pro Ala Lys Leu Cys Ser Asp Asn 290 295 300 Ala Ala Met Ile Ala Gly Leu Gly Gly Glu Asn Phe Gln Lys Asn Ser 305 310 315 320 Ser Ile Pro Glu Ile Arg Ile Cys Ala Arg Tyr Gln Trp Glu Ser Val 325 330 335 Ser Pro Phe Ser Leu Ala Ser Pro 340 7 2060 DNA Chlamydia pneumoniae CDS (101)..(1957) 7 gattttgtgt attttttcag ataatgtttt taaaaaaatg ttttaaaacc ctaaaatcct 60 acctccttgt aaccattctc ggtagaaaag agaggtattt atg aaa aaa ggg aaa 115 Met Lys Lys Gly Lys 1 5 tta gga gcc ata gtt ttt ggc ctt cta ttt aca agt agt gtt gct ggt 163 Leu Gly Ala Ile Val Phe Gly Leu Leu Phe Thr Ser Ser Val Ala Gly 10 15 20 ttt tct aag gat ttg act aaa gac aac gct tat caa gat tta aat gtc 211 Phe Ser Lys Asp Leu Thr Lys Asp Asn Ala Tyr Gln Asp Leu Asn Val 25 30 35 ata gag cat tta ata tcg tta aaa tat gct cct tta cca tgg aag gaa 259 Ile Glu His Leu Ile Ser Leu Lys Tyr Ala Pro Leu Pro Trp Lys Glu 40 45 50 cta tta ttt ggt tgg gat tta tct cag caa aca cag caa gct cgc ttg 307 Leu Leu Phe Gly Trp Asp Leu Ser Gln Gln Thr Gln Gln Ala Arg Leu 55 60 65 caa ctg gtc tta gaa gaa aaa cca aca acc aac tac tgc cag aag gta 355 Gln Leu Val Leu Glu Glu Lys Pro Thr Thr Asn Tyr Cys Gln Lys Val 70 75 80 85 ctc tct aac tac gtg aga tca tta aac gat tat cat gca ggg att acg 403 Leu Ser Asn Tyr Val Arg Ser Leu Asn Asp Tyr His Ala Gly Ile Thr 90 95 100 ttt tat cgt act gaa agt gcg tat atc cct tac gta ttg aag tta agt 451 Phe Tyr Arg Thr Glu Ser Ala Tyr Ile Pro Tyr Val Leu Lys Leu Ser 105 110 115 gaa gat ggt cat gtc ttt gta gtc gac gta cag act agc caa ggg gat 499 Glu Asp Gly His Val Phe Val Val Asp Val Gln Thr Ser Gln Gly Asp 120 125 130 att tac tta ggg gat gaa atc ctt gaa gta gat gga atg ggg att cgt 547 Ile Tyr Leu Gly Asp Glu Ile Leu Glu Val Asp Gly Met Gly Ile Arg 135 140 145 gag gct atc gaa agc ctt cgc ttt gga cga ggg agt gcc aca gac tat 595 Glu Ala Ile Glu Ser Leu Arg Phe Gly Arg Gly Ser Ala Thr Asp Tyr 150 155 160 165 tct gct gca gtt cgt tcc ttg aca tcg cgt tcc gcc gct ttt gga gat 643 Ser Ala Ala Val Arg Ser Leu Thr Ser Arg Ser Ala Ala Phe Gly Asp 170 175 180 gcg gtt cct tca gga att gcc atg ttg aaa ctt cgc cga ccc agt ggt 691 Ala Val Pro Ser Gly Ile Ala Met Leu Lys Leu Arg Arg Pro Ser Gly 185 190 195 ttg atc cgt tcg aca ccg gtc cgt tgg cgt tat act cca gag cat atc 739 Leu Ile Arg Ser Thr Pro Val Arg Trp Arg Tyr Thr Pro Glu His Ile 200 205 210 gga gat ttt tct tta gtt gct cct ttg att cct gaa cat aaa cct caa 787 Gly Asp Phe Ser Leu Val Ala Pro Leu Ile Pro Glu His Lys Pro Gln 215 220 225 tta cct aca caa agt tgt gtg cta ttc cgt tcc ggg gta aat tca cag 835 Leu Pro Thr Gln Ser Cys Val Leu Phe Arg Ser Gly Val Asn Ser Gln 230 235 240 245 tct tct agt agc tct tta ttc agt tcc tac atg gtg cct tat ttc tgg 883 Ser Ser Ser Ser Ser Leu Phe Ser Ser Tyr Met Val Pro Tyr Phe Trp 250 255 260 gaa gaa ttg cgg gtt caa aat aag cag cgt ttt gac agt aat cac cat 931 Glu Glu Leu Arg Val Gln Asn Lys Gln Arg Phe Asp Ser Asn His His 265 270 275 ata ggg agc cgt aat gga ttt tta cct acg ttt ggt cct att ctt tgg 979 Ile Gly Ser Arg Asn Gly Phe Leu Pro Thr Phe Gly Pro Ile Leu Trp 280 285 290 gaa caa gac aag ggg ccc tat cgt tcc tat atc ttt aaa gca aaa gat 1027 Glu Gln Asp Lys Gly Pro Tyr Arg Ser Tyr Ile Phe Lys Ala Lys Asp 295 300 305 tct cag ggc aat ccc cat cgc ata gga ttt tta aga att tct tct tat 1075 Ser Gln Gly Asn Pro His Arg Ile Gly Phe Leu Arg Ile Ser Ser Tyr 310 315 320 325 gtt tgg act gat tta gaa gga ctt gaa gag gat cat aag gat agt cct 1123 Val Trp Thr Asp Leu Glu Gly Leu Glu Glu Asp His Lys Asp Ser Pro 330 335 340 tgg gag ctc ttt gga gag atc atc gat cat ttg gaa aaa gag act gat 1171 Trp Glu Leu Phe Gly Glu Ile Ile Asp His Leu Glu Lys Glu Thr Asp 345 350 355 gct ttg att att gat cag acc cat aat cct gga ggc agt gtt ttc tat 1219 Ala Leu Ile Ile Asp Gln Thr His Asn Pro Gly Gly Ser Val Phe Tyr 360 365 370 ctc tat tcg tta cta tct atg tta aca gat cat cct tta gat act cct 1267 Leu Tyr Ser Leu Leu Ser Met Leu Thr Asp His Pro Leu Asp Thr Pro 375 380 385 aaa cat aga atg att ttc act cag gat gaa gtc agc tcg gct ttg cac 1315 Lys His Arg Met Ile Phe Thr Gln Asp Glu Val Ser Ser Ala Leu His 390 395 400 405 tgg caa gat cta cta gaa gat gtc ttc aca gat gag cag gca gtt gcc 1363 Trp Gln Asp Leu Leu Glu Asp Val Phe Thr Asp Glu Gln Ala Val Ala 410 415 420 gtg cta ggg gaa act atg gaa gga tat tgc atg gat atg cat gct gta 1411 Val Leu Gly Glu Thr Met Glu Gly Tyr Cys Met Asp Met His Ala Val 425 430 435 gcc tct ctt caa aac ttc tct cag agt gtc ctt tct tcc tgg gtt tca 1459 Ala Ser Leu Gln Asn Phe Ser Gln Ser Val Leu Ser Ser Trp Val Ser 440 445 450 ggt gat att aac ctt tca aaa cct atg cct ttg cta gga ttt gca cag 1507 Gly Asp Ile Asn Leu Ser Lys Pro Met Pro Leu Leu Gly Phe Ala Gln 455 460 465 gtt cga cct cat cct aaa cat caa tat act aaa cct ttg ttt atg ttg 1555 Val Arg Pro His Pro Lys His Gln Tyr Thr Lys Pro Leu Phe Met Leu 470 475 480 485 ata gac gag gat gac ttc tct tgt gga gat tta gcg cct gca att ttg 1603 Ile Asp Glu Asp Asp Phe Ser Cys Gly Asp Leu Ala Pro Ala Ile Leu 490 495 500 aag gat aat ggc cgc gct act ctc att gga aag cca aca gca gga gct 1651 Lys Asp Asn Gly Arg Ala Thr Leu Ile Gly Lys Pro Thr Ala Gly Ala 505 510 515 gga ggt ttt gta ttc caa gtc act ttc cct aac cgt tct gga att aaa 1699 Gly Gly Phe Val Phe Gln Val Thr Phe Pro Asn Arg Ser Gly Ile Lys 520 525 530 ggt ctt tct tta aca gga tct tta gct gtt agg aaa gat ggt gag ttt 1747 Gly Leu Ser Leu Thr Gly Ser Leu Ala Val Arg Lys Asp Gly Glu Phe 535 540 545 att gaa aac tta gga gtg gct cct cat att gat tta gga ttt acc tcc 1795 Ile Glu Asn Leu Gly Val Ala Pro His Ile Asp Leu Gly Phe Thr Ser 550 555 560 565 agg gat ttg caa act tcc agg ttt act gat tac gtt gag gca gtg aaa 1843 Arg Asp Leu Gln Thr Ser Arg Phe Thr Asp Tyr Val Glu Ala Val Lys 570 575 580 act ata gtt tta act tct ttg tct gag aac gct aag aag agt gaa gag 1891 Thr Ile Val Leu Thr Ser Leu Ser Glu Asn Ala Lys Lys Ser Glu Glu 585 590 595 cag act tct ccg caa gag acg cct gaa gtt att cga gtc tct tat ccc 1939 Gln Thr Ser Pro Gln Glu Thr Pro Glu Val Ile Arg Val Ser Tyr Pro 600 605 610 aca acg act tct gct tcg taaacgggac gtaatagaat aatttttatt 1987 Thr Thr Thr Ser Ala Ser 615 attgctttaa tatgcgcgct tccaatataa gcattgtgaa gcgcgtttca tatgtctttt 2047 atctttaggt aat 2060 8 619 PRT Chlamydia pneumoniae SITE (328)...(343) B-cell epitope 8 Met Lys Lys Gly Lys Leu Gly Ala Ile Val Phe Gly Leu Leu Phe Thr 1 5 10 15 Ser Ser Val Ala Gly Phe Ser Lys Asp Leu Thr Lys Asp Asn Ala Tyr 20 25 30 Gln Asp Leu Asn Val Ile Glu His Leu Ile Ser Leu Lys Tyr Ala Pro 35 40 45 Leu Pro Trp Lys Glu Leu Leu Phe Gly Trp Asp Leu Ser Gln Gln Thr 50 55 60 Gln Gln Ala Arg Leu Gln Leu Val Leu Glu Glu Lys Pro Thr Thr Asn 65 70 75 80 Tyr Cys Gln Lys Val Leu Ser Asn Tyr Val Arg Ser Leu Asn Asp Tyr 85 90 95 His Ala Gly Ile Thr Phe Tyr Arg Thr Glu Ser Ala Tyr Ile Pro Tyr 100 105 110 Val Leu Lys Leu Ser Glu Asp Gly His Val Phe Val Val Asp Val Gln 115 120 125 Thr Ser Gln Gly Asp Ile Tyr Leu Gly Asp Glu Ile Leu Glu Val Asp 130 135 140 Gly Met Gly Ile Arg Glu Ala Ile Glu Ser Leu Arg Phe Gly Arg Gly 145 150 155 160 Ser Ala Thr Asp Tyr Ser Ala Ala Val Arg Ser Leu Thr Ser Arg Ser 165 170 175 Ala Ala Phe Gly Asp Ala Val Pro Ser Gly Ile Ala Met Leu Lys Leu 180 185 190 Arg Arg Pro Ser Gly Leu Ile Arg Ser Thr Pro Val Arg Trp Arg Tyr 195 200 205 Thr Pro Glu His Ile Gly Asp Phe Ser Leu Val Ala Pro Leu Ile Pro 210 215 220 Glu His Lys Pro Gln Leu Pro Thr Gln Ser Cys Val Leu Phe Arg Ser 225 230 235 240 Gly Val Asn Ser Gln Ser Ser Ser Ser Ser Leu Phe Ser Ser Tyr Met 245 250 255 Val Pro Tyr Phe Trp Glu Glu Leu Arg Val Gln Asn Lys Gln Arg Phe 260 265 270 Asp Ser Asn His His Ile Gly Ser Arg Asn Gly Phe Leu Pro Thr Phe 275 280 285 Gly Pro Ile Leu Trp Glu Gln Asp Lys Gly Pro Tyr Arg Ser Tyr Ile 290 295 300 Phe Lys Ala Lys Asp Ser Gln Gly Asn Pro His Arg Ile Gly Phe Leu 305 310 315 320 Arg Ile Ser Ser Tyr Val Trp Thr Asp Leu Glu Gly Leu Glu Glu Asp 325 330 335 His Lys Asp Ser Pro Trp Glu Leu Phe Gly Glu Ile Ile Asp His Leu 340 345 350 Glu Lys Glu Thr Asp Ala Leu Ile Ile Asp Gln Thr His Asn Pro Gly 355 360 365 Gly Ser Val Phe Tyr Leu Tyr Ser Leu Leu Ser Met Leu Thr Asp His 370 375 380 Pro Leu Asp Thr Pro Lys His Arg Met Ile Phe Thr Gln Asp Glu Val 385 390 395 400 Ser Ser Ala Leu His Trp Gln Asp Leu Leu Glu Asp Val Phe Thr Asp 405 410 415 Glu Gln Ala Val Ala Val Leu Gly Glu Thr Met Glu Gly Tyr Cys Met 420 425 430 Asp Met His Ala Val Ala Ser Leu Gln Asn Phe Ser Gln Ser Val Leu 435 440 445 Ser Ser Trp Val Ser Gly Asp Ile Asn Leu Ser Lys Pro Met Pro Leu 450 455 460 Leu Gly Phe Ala Gln Val Arg Pro His Pro Lys His Gln Tyr Thr Lys 465 470 475 480 Pro Leu Phe Met Leu Ile Asp Glu Asp Asp Phe Ser Cys Gly Asp Leu 485 490 495 Ala Pro Ala Ile Leu Lys Asp Asn Gly Arg Ala Thr Leu Ile Gly Lys 500 505 510 Pro Thr Ala Gly Ala Gly Gly Phe Val Phe Gln Val Thr Phe Pro Asn 515 520 525 Arg Ser Gly Ile Lys Gly Leu Ser Leu Thr Gly Ser Leu Ala Val Arg 530 535 540 Lys Asp Gly Glu Phe Ile Glu Asn Leu Gly Val Ala Pro His Ile Asp 545 550 555 560 Leu Gly Phe Thr Ser Arg Asp Leu Gln Thr Ser Arg Phe Thr Asp Tyr 565 570 575 Val Glu Ala Val Lys Thr Ile Val Leu Thr Ser Leu Ser Glu Asn Ala 580 585 590 Lys Lys Ser Glu Glu Gln Thr Ser Pro Gln Glu Thr Pro Glu Val Ile 595 600 605 Arg Val Ser Tyr Pro Thr Thr Thr Ser Ala Ser 610 615 9 1133 DNA Chlamydia pneumoniae CDS (101)..(1030) 9 gacgtaatag aataattttt attattgctt taatatgcgc gcttccaata taagcattgt 60 gaagcgcgtt tcatatgtct tttatcttta ggtaattatt atg aga aaa ctt att 115 Met Arg Lys Leu Ile 1 5 tta tgc aat cct aga gga ttt tgc tct gga gtt gtg cgc gct att caa 163 Leu Cys Asn Pro Arg Gly Phe Cys Ser Gly Val Val Arg Ala Ile Gln 10 15 20 gtt gta gag gtt gct tta gaa aag tgg gga gct cct atc tat gta aaa 211 Val Val Glu Val Ala Leu Glu Lys Trp Gly Ala Pro Ile Tyr Val Lys 25 30 35 cat gag att gtt cac aat cgc cat gtt gtt aat gct tta cga gcc aag 259 His Glu Ile Val His Asn Arg His Val Val Asn Ala Leu Arg Ala Lys 40 45 50 gga gcg atc ttt gtt gaa gaa ctt gtt gat gtt cct gaa ggt gag aga 307 Gly Ala Ile Phe Val Glu Glu Leu Val Asp Val Pro Glu Gly Glu Arg 55 60 65 gtc att tat tca gct cat gga att cct cct tca gtt aga gct gaa gca 355 Val Ile Tyr Ser Ala His Gly Ile Pro Pro Ser Val Arg Ala Glu Ala 70 75 80 85 aaa gcc cgt aag ctt att gat att gat gct acc tgt ggt ttg gtt act 403 Lys Ala Arg Lys Leu Ile Asp Ile Asp Ala Thr Cys Gly Leu Val Thr 90 95 100 aag gtg cat tct gct gcg aag tta tac gca agt aaa gga tac aaa atc 451 Lys Val His Ser Ala Ala Lys Leu Tyr Ala Ser Lys Gly Tyr Lys Ile 105 110 115 ata ctg atc ggc cat aag aag cac gtt gag gtg att ggt att gtt gga 499 Ile Leu Ile Gly His Lys Lys His Val Glu Val Ile Gly Ile Val Gly 120 125 130 gaa gtt cct gaa cac att act gtt gtc gag aag gtt gct gac gtc gag 547 Glu Val Pro Glu His Ile Thr Val Val Glu Lys Val Ala Asp Val Glu 135 140 145 gcc tta cct ttt agt tct gat aca cct tta ttt tat att act caa acg 595 Ala Leu Pro Phe Ser Ser Asp Thr Pro Leu Phe Tyr Ile Thr Gln Thr 150 155 160 165 acg ttg agt ttg gat gat gtt cag gag atc tca tcg gct ttg cta aag 643 Thr Leu Ser Leu Asp Asp Val Gln Glu Ile Ser Ser Ala Leu Leu Lys 170 175 180 cga tat ccc tct atc att act ctg cct agt tct tcg att tgt tat gca 691 Arg Tyr Pro Ser Ile Ile Thr Leu Pro Ser Ser Ser Ile Cys Tyr Ala 185 190 195 acc acg aac cgt caa aaa gca ttg cgt tct gtt tta tct cgc gtg aat 739 Thr Thr Asn Arg Gln Lys Ala Leu Arg Ser Val Leu Ser Arg Val Asn 200 205 210 tac gtc tat gtg gtt gga gat gtc aac agc tcg aat tcc aat cgt ctt 787 Tyr Val Tyr Val Val Gly Asp Val Asn Ser Ser Asn Ser Asn Arg Leu 215 220 225 cgc gaa gtg gct ttg aga agg gga gtt ccc gct gat ttg atc aac aat 835 Arg Glu Val Ala Leu Arg Arg Gly Val Pro Ala Asp Leu Ile Asn Asn 230 235 240 245 ccc gag gat att gat acg aac atc gta aat cat tct gga gat ata gca 883 Pro Glu Asp Ile Asp Thr Asn Ile Val Asn His Ser Gly Asp Ile Ala 250 255 260 atg act gca gga gcc tca act ccc gaa gac gta gtt caa gct tgc att 931 Met Thr Ala Gly Ala Ser Thr Pro Glu Asp Val Val Gln Ala Cys Ile 265 270 275 cga aag cta tca tca ctt atc cct ggt tta caa gtg gaa aat gat ata 979 Arg Lys Leu Ser Ser Leu Ile Pro Gly Leu Gln Val Glu Asn Asp Ile 280 285 290 ttt gct gta gag gat gtc gta ttt caa tta cca aaa gaa ctc cgt tgt 1027 Phe Ala Val Glu Asp Val Val Phe Gln Leu Pro Lys Glu Leu Arg Cys 295 300 305 tct taggtcttta ggcttacttg ccaagttttt ctcgagattg ctttatagag 1080 Ser 310 tcttcttctc gttcagagag ggtatttacc tttttagttc tctgtatttg aaa 1133 10 310 PRT Chlamydia pneumoniae SITE (198)...(205) B-cell epitope 10 Met Arg Lys Leu Ile Leu Cys Asn Pro Arg Gly Phe Cys Ser Gly Val 1 5 10 15 Val Arg Ala Ile Gln Val Val Glu Val Ala Leu Glu Lys Trp Gly Ala 20 25 30 Pro Ile Tyr Val Lys His Glu Ile Val His Asn Arg His Val Val Asn 35 40 45 Ala Leu Arg Ala Lys Gly Ala Ile Phe Val Glu Glu Leu Val Asp Val 50 55 60 Pro Glu Gly Glu Arg Val Ile Tyr Ser Ala His Gly Ile Pro Pro Ser 65 70 75 80 Val Arg Ala Glu Ala Lys Ala Arg Lys Leu Ile Asp Ile Asp Ala Thr 85 90 95 Cys Gly Leu Val Thr Lys Val His Ser Ala Ala Lys Leu Tyr Ala Ser 100 105 110 Lys Gly Tyr Lys Ile Ile Leu Ile Gly His Lys Lys His Val Glu Val 115 120 125 Ile Gly Ile Val Gly Glu Val Pro Glu His Ile Thr Val Val Glu Lys 130 135 140 Val Ala Asp Val Glu Ala Leu Pro Phe Ser Ser Asp Thr Pro Leu Phe 145 150 155 160 Tyr Ile Thr Gln Thr Thr Leu Ser Leu Asp Asp Val Gln Glu Ile Ser 165 170 175 Ser Ala Leu Leu Lys Arg Tyr Pro Ser Ile Ile Thr Leu Pro Ser Ser 180 185 190 Ser Ile Cys Tyr Ala Thr Thr Asn Arg Gln Lys Ala Leu Arg Ser Val 195 200 205 Leu Ser Arg Val Asn Tyr Val Tyr Val Val Gly Asp Val Asn Ser Ser 210 215 220 Asn Ser Asn Arg Leu Arg Glu Val Ala Leu Arg Arg Gly Val Pro Ala 225 230 235 240 Asp Leu Ile Asn Asn Pro Glu Asp Ile Asp Thr Asn Ile Val Asn His 245 250 255 Ser Gly Asp Ile Ala Met Thr Ala Gly Ala Ser Thr Pro Glu Asp Val 260 265 270 Val Gln Ala Cys Ile Arg Lys Leu Ser Ser Leu Ile Pro Gly Leu Gln 275 280 285 Val Glu Asn Asp Ile Phe Ala Val Glu Asp Val Val Phe Gln Leu Pro 290 295 300 Lys Glu Leu Arg Cys Ser 305 310 11 1466 DNA Chlamydia pneumoniae CDS (101)..(1363) 11 catgggagcc gaggaagcca tctcctacgg acttattgat aaggtggtaa cttctgcgaa 60 agaaactaat aaggatacaa gtagcactta gagagaacat atg aat aaa aaa aat 115 Met Asn Lys Lys Asn 1 5 cta act att tgt tca ttt tgc ggt cgg tct gaa aaa gat gta gag aaa 163 Leu Thr Ile Cys Ser Phe Cys Gly Arg Ser Glu Lys Asp Val Glu Lys 10 15 20 ctg att gct ggg cct tcg gta tac att tgt gac tac tgc atc aaa tta 211 Leu Ile Ala Gly Pro Ser Val Tyr Ile Cys Asp Tyr Cys Ile Lys Leu 25 30 35 tgc tct gga att tta gat aag aaa ccc tcc tct aca ata tcc tca gct 259 Cys Ser Gly Ile Leu Asp Lys Lys Pro Ser Ser Thr Ile Ser Ser Ala 40 45 50 cca gtt tct gaa aca cct tca cag cct tct gat ctc agg gtg ctt acc 307 Pro Val Ser Glu Thr Pro Ser Gln Pro Ser Asp Leu Arg Val Leu Thr 55 60 65 cct aag gaa atc aaa aag cat att gat gaa tat gtc att ggt cag gaa 355 Pro Lys Glu Ile Lys Lys His Ile Asp Glu Tyr Val Ile Gly Gln Glu 70 75 80 85 aga gct aaa aag aca atc gct gtt gct gtt tat aat cac tat aaa cgt 403 Arg Ala Lys Lys Thr Ile Ala Val Ala Val Tyr Asn His Tyr Lys Arg 90 95 100 ata cgt gct cta cta cat aac aaa cag gta agc tac ggg aaa tct aac 451 Ile Arg Ala Leu Leu His Asn Lys Gln Val Ser Tyr Gly Lys Ser Asn 105 110 115 gtg ctt ctc cta ggc cct aca gga tct gga aaa aca tta att gca aaa 499 Val Leu Leu Leu Gly Pro Thr Gly Ser Gly Lys Thr Leu Ile Ala Lys 120 125 130 aca ttg gca aaa att tta gat gtt ccc ttc acc ata gcc gac gca acg 547 Thr Leu Ala Lys Ile Leu Asp Val Pro Phe Thr Ile Ala Asp Ala Thr 135 140 145 acc cta acg gaa gca ggt tat gtc ggt gaa gat gta gag aac att gtc 595 Thr Leu Thr Glu Ala Gly Tyr Val Gly Glu Asp Val Glu Asn Ile Val 150 155 160 165 tta cgt tta tta caa gct gct gat tac gat gtc gcc cgt gca gaa cga 643 Leu Arg Leu Leu Gln Ala Ala Asp Tyr Asp Val Ala Arg Ala Glu Arg 170 175 180 ggc att atc tat atc gat gaa atc gat aaa att gga agg aca aca gca 691 Gly Ile Ile Tyr Ile Asp Glu Ile Asp Lys Ile Gly Arg Thr Thr Ala 185 190 195 aac gtc tcc att act aga gat gtt tct ggc gaa ggg gtt caa caa gca 739 Asn Val Ser Ile Thr Arg Asp Val Ser Gly Glu Gly Val Gln Gln Ala 200 205 210 ttg tta aaa atc gtt gaa gga acc aca gca aac gtt cct cct aaa gga 787 Leu Leu Lys Ile Val Glu Gly Thr Thr Ala Asn Val Pro Pro Lys Gly 215 220 225 gga cgt aag cat cct aac caa gag tat atc cga gtc aat acg gaa aat 835 Gly Arg Lys His Pro Asn Gln Glu Tyr Ile Arg Val Asn Thr Glu Asn 230 235 240 245 atc tta ttt atc gta ggc gga gcc ttc gtc aac cta gat aag att atc 883 Ile Leu Phe Ile Val Gly Gly Ala Phe Val Asn Leu Asp Lys Ile Ile 250 255 260 gca aag cga ttg ggg aaa act acc ata ggg ttt tct gat gat caa gca 931 Ala Lys Arg Leu Gly Lys Thr Thr Ile Gly Phe Ser Asp Asp Gln Ala 265 270 275 gac ctc tct caa aaa acc aga gac cat cta ctt gct aaa gtt gaa acc 979 Asp Leu Ser Gln Lys Thr Arg Asp His Leu Leu Ala Lys Val Glu Thr 280 285 290 gaa gac ctg att gcc ttc gga atg atc cct gaa ttt gtc gga aga ttc 1027 Glu Asp Leu Ile Ala Phe Gly Met Ile Pro Glu Phe Val Gly Arg Phe 295 300 305 aac tgc att gta aac tgt gaa gag ctt tct ttg gat gag ctt gta gcc 1075 Asn Cys Ile Val Asn Cys Glu Glu Leu Ser Leu Asp Glu Leu Val Ala 310 315 320 325 atc ctt aca gaa cct aca aat gcg att gtg aaa caa tat atg gag cta 1123 Ile Leu Thr Glu Pro Thr Asn Ala Ile Val Lys Gln Tyr Met Glu Leu 330 335 340 ttc gca gaa gaa aac gtc aag tta gtc ttc aaa aaa gaa gcc cta tat 1171 Phe Ala Glu Glu Asn Val Lys Leu Val Phe Lys Lys Glu Ala Leu Tyr 345 350 355 gct ata gca aaa aaa gcc aag caa gca aaa act gga gct cgt gct cta 1219 Ala Ile Ala Lys Lys Ala Lys Gln Ala Lys Thr Gly Ala Arg Ala Leu 360 365 370 ggg atg atc cta gaa aat ctc ctt aga gac ctt atg ttt gaa att cct 1267 Gly Met Ile Leu Glu Asn Leu Leu Arg Asp Leu Met Phe Glu Ile Pro 375 380 385 tca gat cct aca gta gaa gct att cat atc caa gaa gac act atc gca 1315 Ser Asp Pro Thr Val Glu Ala Ile His Ile Gln Glu Asp Thr Ile Ala 390 395 400 405 gaa aat aaa gcg cca ata att atc aga agg acc cca gaa gct atc gct 1363 Glu Asn Lys Ala Pro Ile Ile Ile Arg Arg Thr Pro Glu Ala Ile Ala 410 415 420 tagctctttt tagttcctat tttaggggtg tcatgacaac aattgccata gaagctgcaa 1423 aaaaagttct tatcaaacta cgtaatgcag gatatcaggc ata 1466 12 421 PRT Chlamydia pneumoniae SITE (226)...(239) B-cell epitope 12 Met Asn Lys Lys Asn Leu Thr Ile Cys Ser Phe Cys Gly Arg Ser Glu 1 5 10 15 Lys Asp Val Glu Lys Leu Ile Ala Gly Pro Ser Val Tyr Ile Cys Asp 20 25 30 Tyr Cys Ile Lys Leu Cys Ser Gly Ile Leu Asp Lys Lys Pro Ser Ser 35 40 45 Thr Ile Ser Ser Ala Pro Val Ser Glu Thr Pro Ser Gln Pro Ser Asp 50 55 60 Leu Arg Val Leu Thr Pro Lys Glu Ile Lys Lys His Ile Asp Glu Tyr 65 70 75 80 Val Ile Gly Gln Glu Arg Ala Lys Lys Thr Ile Ala Val Ala Val Tyr 85 90 95 Asn His Tyr Lys Arg Ile Arg Ala Leu Leu His Asn Lys Gln Val Ser 100 105 110 Tyr Gly Lys Ser Asn Val Leu Leu Leu Gly Pro Thr Gly Ser Gly Lys 115 120 125 Thr Leu Ile Ala Lys Thr Leu Ala Lys Ile Leu Asp Val Pro Phe Thr 130 135 140 Ile Ala Asp Ala Thr Thr Leu Thr Glu Ala Gly Tyr Val Gly Glu Asp 145 150 155 160 Val Glu Asn Ile Val Leu Arg Leu Leu Gln Ala Ala Asp Tyr Asp Val 165 170 175 Ala Arg Ala Glu Arg Gly Ile Ile Tyr Ile Asp Glu Ile Asp Lys Ile 180 185 190 Gly Arg Thr Thr Ala Asn Val Ser Ile Thr Arg Asp Val Ser Gly Glu 195 200 205 Gly Val Gln Gln Ala Leu Leu Lys Ile Val Glu Gly Thr Thr Ala Asn 210 215 220 Val Pro Pro Lys Gly Gly Arg Lys His Pro Asn Gln Glu Tyr Ile Arg 225 230 235 240 Val Asn Thr Glu Asn Ile Leu Phe Ile Val Gly Gly Ala Phe Val Asn 245 250 255 Leu Asp Lys Ile Ile Ala Lys Arg Leu Gly Lys Thr Thr Ile Gly Phe 260 265 270 Ser Asp Asp Gln Ala Asp Leu Ser Gln Lys Thr Arg Asp His Leu Leu 275 280 285 Ala Lys Val Glu Thr Glu Asp Leu Ile Ala Phe Gly Met Ile Pro Glu 290 295 300 Phe Val Gly Arg Phe Asn Cys Ile Val Asn Cys Glu Glu Leu Ser Leu 305 310 315 320 Asp Glu Leu Val Ala Ile Leu Thr Glu Pro Thr Asn Ala Ile Val Lys 325 330 335 Gln Tyr Met Glu Leu Phe Ala Glu Glu Asn Val Lys Leu Val Phe Lys 340 345 350 Lys Glu Ala Leu Tyr Ala Ile Ala Lys Lys Ala Lys Gln Ala Lys Thr 355 360 365 Gly Ala Arg Ala Leu Gly Met Ile Leu Glu Asn Leu Leu Arg Asp Leu 370 375 380 Met Phe Glu Ile Pro Ser Asp Pro Thr Val Glu Ala Ile His Ile Gln 385 390 395 400 Glu Asp Thr Ile Ala Glu Asn Lys Ala Pro Ile Ile Ile Arg Arg Thr 405 410 415 Pro Glu Ala Ile Ala 420 13 812 DNA Chlamydia pneumoniae CDS (101)..(709) 13 tgacgtagac agcctaaaaa gtcttagcta cgttcctagg gtcatttcgt gatcgggaac 60 gtatggacac aactgaaaat tatttgatga ggaaacgcaa atg aca ctg gta ccc 115 Met Thr Leu Val Pro 1 5 tat gtt gtc gag gat acg ggc cgt ggt gaa agg gcc atg gat att tac 163 Tyr Val Val Glu Asp Thr Gly Arg Gly Glu Arg Ala Met Asp Ile Tyr 10 15 20 tcc cgt ctt ctg aaa gat cgt att gta atg atc ggt cag gaa atc acg 211 Ser Arg Leu Leu Lys Asp Arg Ile Val Met Ile Gly Gln Glu Ile Thr 25 30 35 gag ccc ctc gca aac aca gta att gcc cag ctc ctt ttc ctc atg tcc 259 Glu Pro Leu Ala Asn Thr Val Ile Ala Gln Leu Leu Phe Leu Met Ser 40 45 50 gaa gat cct aaa aag gat att caa att ttc atc aat tcc cca ggc ggc 307 Glu Asp Pro Lys Lys Asp Ile Gln Ile Phe Ile Asn Ser Pro Gly Gly 55 60 65 tac atc acc gct gga ctg gca atc tat gat acc att cgc ttt tta ggt 355 Tyr Ile Thr Ala Gly Leu Ala Ile Tyr Asp Thr Ile Arg Phe Leu Gly 70 75 80 85 tgt gat gta aat acc tac tgc atc ggt caa gct gca tcc atg gga gcc 403 Cys Asp Val Asn Thr Tyr Cys Ile Gly Gln Ala Ala Ser Met Gly Ala 90 95 100 ctc tta tta tcc gca gga act aaa gga aag cgt cac gct ctt ccc cat 451 Leu Leu Leu Ser Ala Gly Thr Lys Gly Lys Arg His Ala Leu Pro His 105 110 115 agc cgt atg atg atc cac caa cct tct gga ggc att atc gga aca tcc 499 Ser Arg Met Met Ile His Gln Pro Ser Gly Gly Ile Ile Gly Thr Ser 120 125 130 gca gac atc caa ctc caa gca gct gaa att cta aca cta aaa aaa cac 547 Ala Asp Ile Gln Leu Gln Ala Ala Glu Ile Leu Thr Leu Lys Lys His 135 140 145 ctt gcc aat atc ctc tct gaa tgc aca gga caa cct gta gaa aaa att 595 Leu Ala Asn Ile Leu Ser Glu Cys Thr Gly Gln Pro Val Glu Lys Ile 150 155 160 165 ata gaa gat tct gaa cga gat ttc ttc atg gga gcc gag gaa gcc atc 643 Ile Glu Asp Ser Glu Arg Asp Phe Phe Met Gly Ala Glu Glu Ala Ile 170 175 180 tcc tac gga ctt att gat aag gtg gta act tct gcg aaa gaa act aat 691 Ser Tyr Gly Leu Ile Asp Lys Val Val Thr Ser Ala Lys Glu Thr Asn 185 190 195 aag gat aca agt agc act tagagagaac atatgaataa aaaaaatcta 739 Lys Asp Thr Ser Ser Thr 200 actatttgtt cattttgcgg tcggtctgaa aaagatgtag agaaactgat tgctgggcct 799 tcggtataca ttt 812 14 203 PRT Chlamydia pneumoniae SITE (107)...(116) B-cell epitope 14 Met Thr Leu Val Pro Tyr Val Val Glu Asp Thr Gly Arg Gly Glu Arg 1 5 10 15 Ala Met Asp Ile Tyr Ser Arg Leu Leu Lys Asp Arg Ile Val Met Ile 20 25 30 Gly Gln Glu Ile Thr Glu Pro Leu Ala Asn Thr Val Ile Ala Gln Leu 35 40 45 Leu Phe Leu Met Ser Glu Asp Pro Lys Lys Asp Ile Gln Ile Phe Ile 50 55 60 Asn Ser Pro Gly Gly Tyr Ile Thr Ala Gly Leu Ala Ile Tyr Asp Thr 65 70 75 80 Ile Arg Phe Leu Gly Cys Asp Val Asn Thr Tyr Cys Ile Gly Gln Ala 85 90 95 Ala Ser Met Gly Ala Leu Leu Leu Ser Ala Gly Thr Lys Gly Lys Arg 100 105 110 His Ala Leu Pro His Ser Arg Met Met Ile His Gln Pro Ser Gly Gly 115 120 125 Ile Ile Gly Thr Ser Ala Asp Ile Gln Leu Gln Ala Ala Glu Ile Leu 130 135 140 Thr Leu Lys Lys His Leu Ala Asn Ile Leu Ser Glu Cys Thr Gly Gln 145 150 155 160 Pro Val Glu Lys Ile Ile Glu Asp Ser Glu Arg Asp Phe Phe Met Gly 165 170 175 Ala Glu Glu Ala Ile Ser Tyr Gly Leu Ile Asp Lys Val Val Thr Ser 180 185 190 Ala Lys Glu Thr Asn Lys Asp Thr Ser Ser Thr 195 200 15 2162 DNA Chlamydia pneumoniae CDS (101)..(2059) 15 gataaaatag aaagacctga tcatttgatg gaaatagcag ctcttcccga ataccaatat 60 ttggaatatc cctcagaaga aagtatcagt cttttatcct atg agc tac cgt aaa 115 Met Ser Tyr Arg Lys 1 5 cgt tcg act cta att gtt cta gga gtg ttt gct ctt tat gct ctt cta 163 Arg Ser Thr Leu Ile Val Leu Gly Val Phe Ala Leu Tyr Ala Leu Leu 10 15 20 gta ttg cgt tat tat aaa att caa att tgt gaa gga gac cac tgg gcc 211 Val Leu Arg Tyr Tyr Lys Ile Gln Ile Cys Glu Gly Asp His Trp Ala 25 30 35 gca gaa gct ctc ggg caa cac gaa ttt tgt gtc cgt gat cct ttt cga 259 Ala Glu Ala Leu Gly Gln His Glu Phe Cys Val Arg Asp Pro Phe Arg 40 45 50 agg ggc acc ttt ttt gct aac acg aca gta cgt aag gga gac aaa gac 307 Arg Gly Thr Phe Phe Ala Asn Thr Thr Val Arg Lys Gly Asp Lys Asp 55 60 65 ctt cag cag cct ttc gct gtc gat att aca aaa ttt cac ctt tgt gca 355 Leu Gln Gln Pro Phe Ala Val Asp Ile Thr Lys Phe His Leu Cys Ala 70 75 80 85 gat cct tta gct att ccc gaa tgt cat cgt gat gag atc atc caa ggg 403 Asp Pro Leu Ala Ile Pro Glu Cys His Arg Asp Glu Ile Ile Gln Gly 90 95 100 att ctc caa ttt att gag ggg cag acc tac gac gac ctc tcc cta aag 451 Ile Leu Gln Phe Ile Glu Gly Gln Thr Tyr Asp Asp Leu Ser Leu Lys 105 110 115 tta gat aag aaa tct cgg tat tgt aag ctg tat cct tta tta gat gtt 499 Leu Asp Lys Lys Ser Arg Tyr Cys Lys Leu Tyr Pro Leu Leu Asp Val 120 125 130 tct gtc cat gac cgg cta tcc ctt tgg tgg aaa gga tat gca aca aag 547 Ser Val His Asp Arg Leu Ser Leu Trp Trp Lys Gly Tyr Ala Thr Lys 135 140 145 cat cgc tta cca aca aac gcc cta ttt ttt att acg gac tac caa cgc 595 His Arg Leu Pro Thr Asn Ala Leu Phe Phe Ile Thr Asp Tyr Gln Arg 150 155 160 165 tcg tat cct ttt ggg aag ctc ctt gga caa gtt ctc cat acc tta aga 643 Ser Tyr Pro Phe Gly Lys Leu Leu Gly Gln Val Leu His Thr Leu Arg 170 175 180 gaa att aag gat gag aaa aca gga aaa gcc ttt ccc aca ggc ggg atg 691 Glu Ile Lys Asp Glu Lys Thr Gly Lys Ala Phe Pro Thr Gly Gly Met 185 190 195 gag gcg tac ttt aat cat att ctg gaa ggg gac gtt gga gag aga aag 739 Glu Ala Tyr Phe Asn His Ile Leu Glu Gly Asp Val Gly Glu Arg Lys 200 205 210 ctg ttg cgt tct cct ttg aac cgt tta gat acg aat cgt gtt atc aaa 787 Leu Leu Arg Ser Pro Leu Asn Arg Leu Asp Thr Asn Arg Val Ile Lys 215 220 225 ctg cct aaa gat ggc tct gat atc tac ctt acg atc aat cct gtg atc 835 Leu Pro Lys Asp Gly Ser Asp Ile Tyr Leu Thr Ile Asn Pro Val Ile 230 235 240 245 cag acc att gca gag gaa gaa ctc gaa cgg ggc gtg cta gaa gct aaa 883 Gln Thr Ile Ala Glu Glu Glu Leu Glu Arg Gly Val Leu Glu Ala Lys 250 255 260 gcc cag ggg ggt agg ctc att cta atg aac tcc caa aca gga gag att 931 Ala Gln Gly Gly Arg Leu Ile Leu Met Asn Ser Gln Thr Gly Glu Ile 265 270 275 ctt gca ctg gct caa tat ccg ttt ttc gat ccc aca aat tat aag gaa 979 Leu Ala Leu Ala Gln Tyr Pro Phe Phe Asp Pro Thr Asn Tyr Lys Glu 280 285 290 tac ttc aat aac aaa gag cgc atc gaa cat acg aag gta tct ttt gtg 1027 Tyr Phe Asn Asn Lys Glu Arg Ile Glu His Thr Lys Val Ser Phe Val 295 300 305 agc gat gtt ttt gaa ccc ggg tcg atc atg aaa cct ttg act gtg gcg 1075 Ser Asp Val Phe Glu Pro Gly Ser Ile Met Lys Pro Leu Thr Val Ala 310 315 320 325 att gct tta caa gct aac gaa gag gct agc tta aaa tcg cag aaa aag 1123 Ile Ala Leu Gln Ala Asn Glu Glu Ala Ser Leu Lys Ser Gln Lys Lys 330 335 340 att ttt gat cct gaa gaa cct atc gat gtg acc agg aca ctc ttc cct 1171 Ile Phe Asp Pro Glu Glu Pro Ile Asp Val Thr Arg Thr Leu Phe Pro 345 350 355 gga cga aaa gga tct ccg ctt aag gat att tct aga aac tct caa ttg 1219 Gly Arg Lys Gly Ser Pro Leu Lys Asp Ile Ser Arg Asn Ser Gln Leu 360 365 370 aat atg tac atg gct atc cag aaa tct tcg aat gtc tat gta gct cag 1267 Asn Met Tyr Met Ala Ile Gln Lys Ser Ser Asn Val Tyr Val Ala Gln 375 380 385 ctg gct gac cgc atc ata caa tct tta gga gtg gcc tgg tac caa cag 1315 Leu Ala Asp Arg Ile Ile Gln Ser Leu Gly Val Ala Trp Tyr Gln Gln 390 395 400 405 aag ttg cta gct ctg gga ttt gga aga aaa aca ggg atc gag ctt ccc 1363 Lys Leu Leu Ala Leu Gly Phe Gly Arg Lys Thr Gly Ile Glu Leu Pro 410 415 420 agt gag gcc tct ggt ttg gtg cct tct ccc cat cgt ttc cat att aat 1411 Ser Glu Ala Ser Gly Leu Val Pro Ser Pro His Arg Phe His Ile Asn 425 430 435 ggt tcc ctg gaa tgg tcc tta tct act cca tat tct ttg gct atg gga 1459 Gly Ser Leu Glu Trp Ser Leu Ser Thr Pro Tyr Ser Leu Ala Met Gly 440 445 450 tat aat att ttg gca aca ggg ata caa atg gtt caa gcc tac gct atc 1507 Tyr Asn Ile Leu Ala Thr Gly Ile Gln Met Val Gln Ala Tyr Ala Ile 455 460 465 ctt gca aac gga ggt tat gcc gtc cgg ccc act tta gta aaa aag atc 1555 Leu Ala Asn Gly Gly Tyr Ala Val Arg Pro Thr Leu Val Lys Lys Ile 470 475 480 485 gtc tct gct tca gga gag gaa tat cat ctt cct act aaa gag aag aca 1603 Val Ser Ala Ser Gly Glu Glu Tyr His Leu Pro Thr Lys Glu Lys Thr 490 495 500 cga ctc ttt tca gaa gaa att act aga gaa gtt gtt cgt gcc atg cgt 1651 Arg Leu Phe Ser Glu Glu Ile Thr Arg Glu Val Val Arg Ala Met Arg 505 510 515 ttt aca acg tta ccc gga ggt tcg gga ttt cga gcc tct cct aag cat 1699 Phe Thr Thr Leu Pro Gly Gly Ser Gly Phe Arg Ala Ser Pro Lys His 520 525 530 cac tct agt gct ggg aaa aca gga act aca gaa aag atg att cat gga 1747 His Ser Ser Ala Gly Lys Thr Gly Thr Thr Glu Lys Met Ile His Gly 535 540 545 aaa tat gat aaa cgc cgt cat att gct tct ttt ata ggt ttt act ccc 1795 Lys Tyr Asp Lys Arg Arg His Ile Ala Ser Phe Ile Gly Phe Thr Pro 550 555 560 565 gta gag agc tcg gag gga aat ttc cca cct tta gtg atg ctc gtc tcc 1843 Val Glu Ser Ser Glu Gly Asn Phe Pro Pro Leu Val Met Leu Val Ser 570 575 580 ata gat gat cct gaa tat ggt ttg cga gcc gac ggc acg aaa aat tat 1891 Ile Asp Asp Pro Glu Tyr Gly Leu Arg Ala Asp Gly Thr Lys Asn Tyr 585 590 595 atg ggg ggg cgt tgt gcg gca ccc att ttt tct agg gtt gct gac cgc 1939 Met Gly Gly Arg Cys Ala Ala Pro Ile Phe Ser Arg Val Ala Asp Arg 600 605 610 aca ctc ctc tat tta ggg att ctt cca gac aag aag cta aga aat tgc 1987 Thr Leu Leu Tyr Leu Gly Ile Leu Pro Asp Lys Lys Leu Arg Asn Cys 615 620 625 gac gaa gaa gct gct gca tta aag cgt ctc tat gaa gaa tgg aat cgt 2035 Asp Glu Glu Ala Ala Ala Leu Lys Arg Leu Tyr Glu Glu Trp Asn Arg 630 635 640 645 tct ccg aaa caa ggg gga acg agg tgaggatctc tatttccatc ttgctataga 2089 Ser Pro Lys Gln Gly Gly Thr Arg 650 cttttaccgt tgagcaaaga ctctctatca gagagcccgt ctcctcttta tcctctatga 2149 gtagtttatg tta 2162 16 653 PRT Chlamydia pneumoniae SITE (287)...(306) B-cell epitope 16 Met Ser Tyr Arg Lys Arg Ser Thr Leu Ile Val Leu Gly Val Phe Ala 1 5 10 15 Leu Tyr Ala Leu Leu Val Leu Arg Tyr Tyr Lys Ile Gln Ile Cys Glu 20 25 30 Gly Asp His Trp Ala Ala Glu Ala Leu Gly Gln His Glu Phe Cys Val 35 40 45 Arg Asp Pro Phe Arg Arg Gly Thr Phe Phe Ala Asn Thr Thr Val Arg 50 55 60 Lys Gly Asp Lys Asp Leu Gln Gln Pro Phe Ala Val Asp Ile Thr Lys 65 70 75 80 Phe His Leu Cys Ala Asp Pro Leu Ala Ile Pro Glu Cys His Arg Asp 85 90 95 Glu Ile Ile Gln Gly Ile Leu Gln Phe Ile Glu Gly Gln Thr Tyr Asp 100 105 110 Asp Leu Ser Leu Lys Leu Asp Lys Lys Ser Arg Tyr Cys Lys Leu Tyr 115 120 125 Pro Leu Leu Asp Val Ser Val His Asp Arg Leu Ser Leu Trp Trp Lys 130 135 140 Gly Tyr Ala Thr Lys His Arg Leu Pro Thr Asn Ala Leu Phe Phe Ile 145 150 155 160 Thr Asp Tyr Gln Arg Ser Tyr Pro Phe Gly Lys Leu Leu Gly Gln Val 165 170 175 Leu His Thr Leu Arg Glu Ile Lys Asp Glu Lys Thr Gly Lys Ala Phe 180 185 190 Pro Thr Gly Gly Met Glu Ala Tyr Phe Asn His Ile Leu Glu Gly Asp 195 200 205 Val Gly Glu Arg Lys Leu Leu Arg Ser Pro Leu Asn Arg Leu Asp Thr 210 215 220 Asn Arg Val Ile Lys Leu Pro Lys Asp Gly Ser Asp Ile Tyr Leu Thr 225 230 235 240 Ile Asn Pro Val Ile Gln Thr Ile Ala Glu Glu Glu Leu Glu Arg Gly 245 250 255 Val Leu Glu Ala Lys Ala Gln Gly Gly Arg Leu Ile Leu Met Asn Ser 260 265 270 Gln Thr Gly Glu Ile Leu Ala Leu Ala Gln Tyr Pro Phe Phe Asp Pro 275 280 285 Thr Asn Tyr Lys Glu Tyr Phe Asn Asn Lys Glu Arg Ile Glu His Thr 290 295 300 Lys Val Ser Phe Val Ser Asp Val Phe Glu Pro Gly Ser Ile Met Lys 305 310 315 320 Pro Leu Thr Val Ala Ile Ala Leu Gln Ala Asn Glu Glu Ala Ser Leu 325 330 335 Lys Ser Gln Lys Lys Ile Phe Asp Pro Glu Glu Pro Ile Asp Val Thr 340 345 350 Arg Thr Leu Phe Pro Gly Arg Lys Gly Ser Pro Leu Lys Asp Ile Ser 355 360 365 Arg Asn Ser Gln Leu Asn Met Tyr Met Ala Ile Gln Lys Ser Ser Asn 370 375 380 Val Tyr Val Ala Gln Leu Ala Asp Arg Ile Ile Gln Ser Leu Gly Val 385 390 395 400 Ala Trp Tyr Gln Gln Lys Leu Leu Ala Leu Gly Phe Gly Arg Lys Thr 405 410 415 Gly Ile Glu Leu Pro Ser Glu Ala Ser Gly Leu Val Pro Ser Pro His 420 425 430 Arg Phe His Ile Asn Gly Ser Leu Glu Trp Ser Leu Ser Thr Pro Tyr 435 440 445 Ser Leu Ala Met Gly Tyr Asn Ile Leu Ala Thr Gly Ile Gln Met Val 450 455 460 Gln Ala Tyr Ala Ile Leu Ala Asn Gly Gly Tyr Ala Val Arg Pro Thr 465 470 475 480 Leu Val Lys Lys Ile Val Ser Ala Ser Gly Glu Glu Tyr His Leu Pro 485 490 495 Thr Lys Glu Lys Thr Arg Leu Phe Ser Glu Glu Ile Thr Arg Glu Val 500 505 510 Val Arg Ala Met Arg Phe Thr Thr Leu Pro Gly Gly Ser Gly Phe Arg 515 520 525 Ala Ser Pro Lys His His Ser Ser Ala Gly Lys Thr Gly Thr Thr Glu 530 535 540 Lys Met Ile His Gly Lys Tyr Asp Lys Arg Arg His Ile Ala Ser Phe 545 550 555 560 Ile Gly Phe Thr Pro Val Glu Ser Ser Glu Gly Asn Phe Pro Pro Leu 565 570 575 Val Met Leu Val Ser Ile Asp Asp Pro Glu Tyr Gly Leu Arg Ala Asp 580 585 590 Gly Thr Lys Asn Tyr Met Gly Gly Arg Cys Ala Ala Pro Ile Phe Ser 595 600 605 Arg Val Ala Asp Arg Thr Leu Leu Tyr Leu Gly Ile Leu Pro Asp Lys 610 615 620 Lys Leu Arg Asn Cys Asp Glu Glu Ala Ala Ala Leu Lys Arg Leu Tyr 625 630 635 640 Glu Glu Trp Asn Arg Ser Pro Lys Gln Gly Gly Thr Arg 645 650 17 2738 DNA Chlamydia pneumoniae CDS (101)..(2635) 17 gaattttacc aaatttgctg gtttagagcg aagagttgca tcattatttt aaatttcgta 60 tatgcttaag gaaagttcta cccctgtctt ttaggttttt atg ttt gag aag ttc 115 Met Phe Glu Lys Phe 1 5 act aat aga gca aaa caa gtc att aaa ctg gcg aaa aag gag gct cag 163 Thr Asn Arg Ala Lys Gln Val Ile Lys Leu Ala Lys Lys Glu Ala Gln 10 15 20 cgt tta aat cat aac tac ctg ggt act gag cac atc ctg ctt ggt ctt 211 Arg Leu Asn His Asn Tyr Leu Gly Thr Glu His Ile Leu Leu Gly Leu 25 30 35 ctc aaa ctt ggt caa ggg gta gct gtt aat gta tta cgc aac ctc ggt 259 Leu Lys Leu Gly Gln Gly Val Ala Val Asn Val Leu Arg Asn Leu Gly 40 45 50 ata gat ttt gat acg gca cgg caa gag gtg gaa cgc ctg att ggt tat 307 Ile Asp Phe Asp Thr Ala Arg Gln Glu Val Glu Arg Leu Ile Gly Tyr 55 60 65 ggt cca gaa att caa gtc tac gga gac cct gcc ctt aca gga aga gta 355 Gly Pro Glu Ile Gln Val Tyr Gly Asp Pro Ala Leu Thr Gly Arg Val 70 75 80 85 aaa aaa tct ttt gaa tca gca aat gaa gag gcc agc ctt tta gag cac 403 Lys Lys Ser Phe Glu Ser Ala Asn Glu Glu Ala Ser Leu Leu Glu His 90 95 100 aat tat gtc ggg acg gag cat tta ctc tta ggg atc cta cat caa tca 451 Asn Tyr Val Gly Thr Glu His Leu Leu Leu Gly Ile Leu His Gln Ser 105 110 115 gat agt gtc gct ctt cag gta tta gaa aac tta cat atc gat cca aga 499 Asp Ser Val Ala Leu Gln Val Leu Glu Asn Leu His Ile Asp Pro Arg 120 125 130 gag gtt cgt aag gaa att ctt aga gaa tta gag acc ttc aat cta caa 547 Glu Val Arg Lys Glu Ile Leu Arg Glu Leu Glu Thr Phe Asn Leu Gln 135 140 145 ctt cct cct tcg tcg tcg tct tct tcc tca tcc tct cga agc aac cct 595 Leu Pro Pro Ser Ser Ser Ser Ser Ser Ser Ser Ser Arg Ser Asn Pro 150 155 160 165 tca tct tca aaa tct cct tta ggt cat agc tta ggt tct gac aaa aac 643 Ser Ser Ser Lys Ser Pro Leu Gly His Ser Leu Gly Ser Asp Lys Asn 170 175 180 gaa aag ctt tct gct ctg aaa gca tat ggt tat gat tta acg gag atg 691 Glu Lys Leu Ser Ala Leu Lys Ala Tyr Gly Tyr Asp Leu Thr Glu Met 185 190 195 gtc cga gag tct aag ctc gat cct gtc att ggt cgt tct tca gaa gtc 739 Val Arg Glu Ser Lys Leu Asp Pro Val Ile Gly Arg Ser Ser Glu Val 200 205 210 gaa cgg ttg att ttg att ctt tgc cga aga aga aaa aac aat cct gta 787 Glu Arg Leu Ile Leu Ile Leu Cys Arg Arg Arg Lys Asn Asn Pro Val 215 220 225 ctt att gga gaa gct gga gtt ggt aag act gca att gtt gag ggt ctg 835 Leu Ile Gly Glu Ala Gly Val Gly Lys Thr Ala Ile Val Glu Gly Leu 230 235 240 245 gct caa aaa atc att ctg aat gag gtt cct gat gcc tta cgg aaa aag 883 Ala Gln Lys Ile Ile Leu Asn Glu Val Pro Asp Ala Leu Arg Lys Lys 250 255 260 cga ctg att act cta gat cta gca tta atg att gct gga aca aaa tat 931 Arg Leu Ile Thr Leu Asp Leu Ala Leu Met Ile Ala Gly Thr Lys Tyr 265 270 275 cga ggg caa ttt gag gaa cgg atc aaa gct gtc atg gat gaa gtt cgc 979 Arg Gly Gln Phe Glu Glu Arg Ile Lys Ala Val Met Asp Glu Val Arg 280 285 290 aag cat gga aac atc ttg ctc ttc att gac gag ctc cac acg att gta 1027 Lys His Gly Asn Ile Leu Leu Phe Ile Asp Glu Leu His Thr Ile Val 295 300 305 gga gca gga gca gct gaa ggt gct atc gat gct tca aac att tta aaa 1075 Gly Ala Gly Ala Ala Glu Gly Ala Ile Asp Ala Ser Asn Ile Leu Lys 310 315 320 325 cct gcg tta gcg cga ggt gaa att cag tgt att gga gca act acg ata 1123 Pro Ala Leu Ala Arg Gly Glu Ile Gln Cys Ile Gly Ala Thr Thr Ile 330 335 340 gat gag tat cgc aag cac ata gaa aaa gac gca gct tta gaa cgt cgt 1171 Asp Glu Tyr Arg Lys His Ile Glu Lys Asp Ala Ala Leu Glu Arg Arg 345 350 355 ttc caa aaa atc gtg gtt cac cct cct agt gta gat gag act att gag 1219 Phe Gln Lys Ile Val Val His Pro Pro Ser Val Asp Glu Thr Ile Glu 360 365 370 att tta cgt ggc ctc aag aaa aag tat gaa gaa cat cac aat gtc ttc 1267 Ile Leu Arg Gly Leu Lys Lys Lys Tyr Glu Glu His His Asn Val Phe 375 380 385 att act gaa gaa gct tta aaa gca gct gcg act ctt tct gat caa tat 1315 Ile Thr Glu Glu Ala Leu Lys Ala Ala Ala Thr Leu Ser Asp Gln Tyr 390 395 400 405 gtt cat gga cgt ttc ctc cct gat aaa gca ata gat ctt tta gat gaa 1363 Val His Gly Arg Phe Leu Pro Asp Lys Ala Ile Asp Leu Leu Asp Glu 410 415 420 gct ggg gct cgt gtc cgt gtg aat aca atg ggt cag cct aca gat tta 1411 Ala Gly Ala Arg Val Arg Val Asn Thr Met Gly Gln Pro Thr Asp Leu 425 430 435 atg aag cta gag gct gaa atc gaa aat aca aaa ttg gcc aaa gag cag 1459 Met Lys Leu Glu Ala Glu Ile Glu Asn Thr Lys Leu Ala Lys Glu Gln 440 445 450 gcc att gga act caa gaa tac gaa aaa gct gca ggt tta cgt gat gaa 1507 Ala Ile Gly Thr Gln Glu Tyr Glu Lys Ala Ala Gly Leu Arg Asp Glu 455 460 465 gag aaa aaa ctt cgc gaa cgt ctg caa agt atg aaa cag gaa tgg gaa 1555 Glu Lys Lys Leu Arg Glu Arg Leu Gln Ser Met Lys Gln Glu Trp Glu 470 475 480 485 aat cat aaa gaa gag cac caa gtt cct gta gat gaa gaa gca gtc gct 1603 Asn His Lys Glu Glu His Gln Val Pro Val Asp Glu Glu Ala Val Ala 490 495 500 cag gta gtt tct cta caa aca gga att ccc tca gca agg ctc aca gaa 1651 Gln Val Val Ser Leu Gln Thr Gly Ile Pro Ser Ala Arg Leu Thr Glu 505 510 515 gct gaa agt gag aag ctt ctg aag tta gaa gac acg tta aga aga aaa 1699 Ala Glu Ser Glu Lys Leu Leu Lys Leu Glu Asp Thr Leu Arg Arg Lys 520 525 530 gtc att ggt caa aat gat gcc gtt acc agc att tgc cgt gcc atc cga 1747 Val Ile Gly Gln Asn Asp Ala Val Thr Ser Ile Cys Arg Ala Ile Arg 535 540 545 cgt tct cga aca ggg atc aaa gat cct aac cga cct acg ggc tcc ttc 1795 Arg Ser Arg Thr Gly Ile Lys Asp Pro Asn Arg Pro Thr Gly Ser Phe 550 555 560 565 cta ttc ctt ggg cct acc ggt gta ggg aaa agc ctg ctc gcc caa caa 1843 Leu Phe Leu Gly Pro Thr Gly Val Gly Lys Ser Leu Leu Ala Gln Gln 570 575 580 att gct ata gag atg ttc ggt ggt gaa gac gct ctg att cag gta gac 1891 Ile Ala Ile Glu Met Phe Gly Gly Glu Asp Ala Leu Ile Gln Val Asp 585 590 595 atg tca gag tac atg gag aaa ttt gct gct acc aag atg atg gga tca 1939 Met Ser Glu Tyr Met Glu Lys Phe Ala Ala Thr Lys Met Met Gly Ser 600 605 610 cct cca gga tat gta ggt cat gaa gaa ggg ggc cac ctt acg gaa cag 1987 Pro Pro Gly Tyr Val Gly His Glu Glu Gly Gly His Leu Thr Glu Gln 615 620 625 gta cgt cgc cgt cct tac tgc gtt gtt ctc ttt gat gag ata gaa aag 2035 Val Arg Arg Arg Pro Tyr Cys Val Val Leu Phe Asp Glu Ile Glu Lys 630 635 640 645 gca cac cca gac att atg gac ctg atg ttg caa att tta gag caa gga 2083 Ala His Pro Asp Ile Met Asp Leu Met Leu Gln Ile Leu Glu Gln Gly 650 655 660 cgt ctt act gat tct ttt ggt cgc aaa gtg gat ttc cgt cat gcc att 2131 Arg Leu Thr Asp Ser Phe Gly Arg Lys Val Asp Phe Arg His Ala Ile 665 670 675 att atc atg acc tcc aat ttg gga gct gat ctc att cgt aaa agc gga 2179 Ile Ile Met Thr Ser Asn Leu Gly Ala Asp Leu Ile Arg Lys Ser Gly 680 685 690 gaa att ggt ttt ggc ttg aag tcc cat atg gac tat aag gtc atc caa 2227 Glu Ile Gly Phe Gly Leu Lys Ser His Met Asp Tyr Lys Val Ile Gln 695 700 705 gag aaa atc gaa cat gct atg aag aaa cac tta aag cct gag ttc att 2275 Glu Lys Ile Glu His Ala Met Lys Lys His Leu Lys Pro Glu Phe Ile 710 715 720 725 aac cgt ttg gat gaa agt gtg att ttc cgt ccc ctc gag aaa gaa tct 2323 Asn Arg Leu Asp Glu Ser Val Ile Phe Arg Pro Leu Glu Lys Glu Ser 730 735 740 cta tcg gag atc atc cat tta gag atc aac aaa ctg gac tcg aga ctg 2371 Leu Ser Glu Ile Ile His Leu Glu Ile Asn Lys Leu Asp Ser Arg Leu 745 750 755 aaa aac tac caa atg gct ttg aac atc cca gac tct gtg att tcc ttc 2419 Lys Asn Tyr Gln Met Ala Leu Asn Ile Pro Asp Ser Val Ile Ser Phe 760 765 770 cta gta acg aag ggg cat tct cca gaa atg gga gca cgt cct cta cgc 2467 Leu Val Thr Lys Gly His Ser Pro Glu Met Gly Ala Arg Pro Leu Arg 775 780 785 cgt gtc att gag cag tac ctt gaa gat cct cta gcg gag ctc ttg ctt 2515 Arg Val Ile Glu Gln Tyr Leu Glu Asp Pro Leu Ala Glu Leu Leu Leu 790 795 800 805 aaa gag tcc tgc cgt caa gaa gct cgc aag cta cga gca acc ttg gtt 2563 Lys Glu Ser Cys Arg Gln Glu Ala Arg Lys Leu Arg Ala Thr Leu Val 810 815 820 gaa aat cgc gtt gcc ttt gaa agg gaa gaa gag gag cag gaa gct gct 2611 Glu Asn Arg Val Ala Phe Glu Arg Glu Glu Glu Glu Gln Glu Ala Ala 825 830 835 ctc cct agc cct cac ttg gaa tca taggaacgtc gataactcca ctaccaaggc 2665 Leu Pro Ser Pro His Leu Glu Ser 840 845 aggtatctcc ttgataaaac gctattgttt gtcctggagt taccgccttg acgggttgtg 2725 aaaatcgcac ctt 2738 18 845 PRT Chlamydia pneumoniae SITE (467)...(492) B-cell epitope 18 Met Phe Glu Lys Phe Thr Asn Arg Ala Lys Gln Val Ile Lys Leu Ala 1 5 10 15 Lys Lys Glu Ala Gln Arg Leu Asn His Asn Tyr Leu Gly Thr Glu His 20 25 30 Ile Leu Leu Gly Leu Leu Lys Leu Gly Gln Gly Val Ala Val Asn Val 35 40 45 Leu Arg Asn Leu Gly Ile Asp Phe Asp Thr Ala Arg Gln Glu Val Glu 50 55 60 Arg Leu Ile Gly Tyr Gly Pro Glu Ile Gln Val Tyr Gly Asp Pro Ala 65 70 75 80 Leu Thr Gly Arg Val Lys Lys Ser Phe Glu Ser Ala Asn Glu Glu Ala 85 90 95 Ser Leu Leu Glu His Asn Tyr Val Gly Thr Glu His Leu Leu Leu Gly 100 105 110 Ile Leu His Gln Ser Asp Ser Val Ala Leu Gln Val Leu Glu Asn Leu 115 120 125 His Ile Asp Pro Arg Glu Val Arg Lys Glu Ile Leu Arg Glu Leu Glu 130 135 140 Thr Phe Asn Leu Gln Leu Pro Pro Ser Ser Ser Ser Ser Ser Ser Ser 145 150 155 160 Ser Arg Ser Asn Pro Ser Ser Ser Lys Ser Pro Leu Gly His Ser Leu 165 170 175 Gly Ser Asp Lys Asn Glu Lys Leu Ser Ala Leu Lys Ala Tyr Gly Tyr 180 185 190 Asp Leu Thr Glu Met Val Arg Glu Ser Lys Leu Asp Pro Val Ile Gly 195 200 205 Arg Ser Ser Glu Val Glu Arg Leu Ile Leu Ile Leu Cys Arg Arg Arg 210 215 220 Lys Asn Asn Pro Val Leu Ile Gly Glu Ala Gly Val Gly Lys Thr Ala 225 230 235 240 Ile Val Glu Gly Leu Ala Gln Lys Ile Ile Leu Asn Glu Val Pro Asp 245 250 255 Ala Leu Arg Lys Lys Arg Leu Ile Thr Leu Asp Leu Ala Leu Met Ile 260 265 270 Ala Gly Thr Lys Tyr Arg Gly Gln Phe Glu Glu Arg Ile Lys Ala Val 275 280 285 Met Asp Glu Val Arg Lys His Gly Asn Ile Leu Leu Phe Ile Asp Glu 290 295 300 Leu His Thr Ile Val Gly Ala Gly Ala Ala Glu Gly Ala Ile Asp Ala 305 310 315 320 Ser Asn Ile Leu Lys Pro Ala Leu Ala Arg Gly Glu Ile Gln Cys Ile 325 330 335 Gly Ala Thr Thr Ile Asp Glu Tyr Arg Lys His Ile Glu Lys Asp Ala 340 345 350 Ala Leu Glu Arg Arg Phe Gln Lys Ile Val Val His Pro Pro Ser Val 355 360 365 Asp Glu Thr Ile Glu Ile Leu Arg Gly Leu Lys Lys Lys Tyr Glu Glu 370 375 380 His His Asn Val Phe Ile Thr Glu Glu Ala Leu Lys Ala Ala Ala Thr 385 390 395 400 Leu Ser Asp Gln Tyr Val His Gly Arg Phe Leu Pro Asp Lys Ala Ile 405 410 415 Asp Leu Leu Asp Glu Ala Gly Ala Arg Val Arg Val Asn Thr Met Gly 420 425 430 Gln Pro Thr Asp Leu Met Lys Leu Glu Ala Glu Ile Glu Asn Thr Lys 435 440 445 Leu Ala Lys Glu Gln Ala Ile Gly Thr Gln Glu Tyr Glu Lys Ala Ala 450 455 460 Gly Leu Arg Asp Glu Glu Lys Lys Leu Arg Glu Arg Leu Gln Ser Met 465 470 475 480 Lys Gln Glu Trp Glu Asn His Lys Glu Glu His Gln Val Pro Val Asp 485 490 495 Glu Glu Ala Val Ala Gln Val Val Ser Leu Gln Thr Gly Ile Pro Ser 500 505 510 Ala Arg Leu Thr Glu Ala Glu Ser Glu Lys Leu Leu Lys Leu Glu Asp 515 520 525 Thr Leu Arg Arg Lys Val Ile Gly Gln Asn Asp Ala Val Thr Ser Ile 530 535 540 Cys Arg Ala Ile Arg Arg Ser Arg Thr Gly Ile Lys Asp Pro Asn Arg 545 550 555 560 Pro Thr Gly Ser Phe Leu Phe Leu Gly Pro Thr Gly Val Gly Lys Ser 565 570 575 Leu Leu Ala Gln Gln Ile Ala Ile Glu Met Phe Gly Gly Glu Asp Ala 580 585 590 Leu Ile Gln Val Asp Met Ser Glu Tyr Met Glu Lys Phe Ala Ala Thr 595 600 605 Lys Met Met Gly Ser Pro Pro Gly Tyr Val Gly His Glu Glu Gly Gly 610 615 620 His Leu Thr Glu Gln Val Arg Arg Arg Pro Tyr Cys Val Val Leu Phe 625 630 635 640 Asp Glu Ile Glu Lys Ala His Pro Asp Ile Met Asp Leu Met Leu Gln 645 650 655 Ile Leu Glu Gln Gly Arg Leu Thr Asp Ser Phe Gly Arg Lys Val Asp 660 665 670 Phe Arg His Ala Ile Ile Ile Met Thr Ser Asn Leu Gly Ala Asp Leu 675 680 685 Ile Arg Lys Ser Gly Glu Ile Gly Phe Gly Leu Lys Ser His Met Asp 690 695 700 Tyr Lys Val Ile Gln Glu Lys Ile Glu His Ala Met Lys Lys His Leu 705 710 715 720 Lys Pro Glu Phe Ile Asn Arg Leu Asp Glu Ser Val Ile Phe Arg Pro 725 730 735 Leu Glu Lys Glu Ser Leu Ser Glu Ile Ile His Leu Glu Ile Asn Lys 740 745 750 Leu Asp Ser Arg Leu Lys Asn Tyr Gln Met Ala Leu Asn Ile Pro Asp 755 760 765 Ser Val Ile Ser Phe Leu Val Thr Lys Gly His Ser Pro Glu Met Gly 770 775 780 Ala Arg Pro Leu Arg Arg Val Ile Glu Gln Tyr Leu Glu Asp Pro Leu 785 790 795 800 Ala Glu Leu Leu Leu Lys Glu Ser Cys Arg Gln Glu Ala Arg Lys Leu 805 810 815 Arg Ala Thr Leu Val Glu Asn Arg Val Ala Phe Glu Arg Glu Glu Glu 820 825 830 Glu Gln Glu Ala Ala Leu Pro Ser Pro His Leu Glu Ser 835 840 845 19 509 DNA Chlamydia pneumoniae CDS (101)..(406) 19 gattcaggtt ctagtgagct tatgctcatg gaagttcaag tcttcttagc tgcaagaaaa 60 taacagggac agtaattcga tttttcgaga agggaaactt atg gta aag atc ata 115 Met Val Lys Ile Ile 1 5 tca agt gaa aat ttt gac tct ttt att gca tcg ggg ctc gtt ctc gtt 163 Ser Ser Glu Asn Phe Asp Ser Phe Ile Ala Ser Gly Leu Val Leu Val 10 15 20 gat ttc ttt gca gaa tgg tgt ggc ccc tgt cgg atg ctc act cct atc 211 Asp Phe Phe Ala Glu Trp Cys Gly Pro Cys Arg Met Leu Thr Pro Ile 25 30 35 tta gaa aat ctt gct gcg gaa ctt cct cat gtc act att gga aaa atc 259 Leu Glu Asn Leu Ala Ala Glu Leu Pro His Val Thr Ile Gly Lys Ile 40 45 50 aat ata gat gag aac agc aag cct gca gaa acg tac gaa gtc agc tct 307 Asn Ile Asp Glu Asn Ser Lys Pro Ala Glu Thr Tyr Glu Val Ser Ser 55 60 65 att cct acg ctt att ctt ttt aag gat ggg aac gag gtg gct cgg gtc 355 Ile Pro Thr Leu Ile Leu Phe Lys Asp Gly Asn Glu Val Ala Arg Val 70 75 80 85 gta ggt ctt aag gat aaa gaa ttc cta acc aat ctt atc aat aag cac 403 Val Gly Leu Lys Asp Lys Glu Phe Leu Thr Asn Leu Ile Asn Lys His 90 95 100 gct taaaaagacg ctgcaatatt aaaccgtagg attcttttgc aatgctacgg 456 Ala ttttctgcct taccacttca tataaaacga tccctacact ggtagctaaa ttt 509 20 102 PRT Chlamydia pneumoniae SITE (543)...(66) B-cell epitope 20 Met Val Lys Ile Ile Ser Ser Glu Asn Phe Asp Ser Phe Ile Ala Ser 1 5 10 15 Gly Leu Val Leu Val Asp Phe Phe Ala Glu Trp Cys Gly Pro Cys Arg 20 25 30 Met Leu Thr Pro Ile Leu Glu Asn Leu Ala Ala Glu Leu Pro His Val 35 40 45 Thr Ile Gly Lys Ile Asn Ile Asp Glu Asn Ser Lys Pro Ala Glu Thr 50 55 60 Tyr Glu Val Ser Ser Ile Pro Thr Leu Ile Leu Phe Lys Asp Gly Asn 65 70 75 80 Glu Val Ala Arg Val Val Gly Leu Lys Asp Lys Glu Phe Leu Thr Asn 85 90 95 Leu Ile Asn Lys His Ala 100 21 43 DNA Artificial Sequence 5′ PCR primer 21 ataagaatgc ggccgccacc atgaagatgc ataggcttaa acc 43 22 36 DNA Artificial Sequence 3′ PCR primer 22 gcgccggatc ccacttaaga tatcgatatt tttgag 36 23 45 DNA Artificial Sequence 5′ PCR primer 23 ataagaatgc ggccgccacc atgcggttgg gaaataagcc tatgc 45 24 35 DNA Artificial Sequence 3′ PCR primer 24 gcgccggtac cgtaatttaa tactctttga agggc 35 25 49 DNA Artificial Sequence 5′ PCR primer 25 ataagaatgc ggccgccacc atgctcaccc taggcttgga aagttcttg 49 26 36 DNA Artificial Sequence 3′ PCR primer 26 gctttggagg atccccggag aggctaagga gaatgg 36 27 44 DNA Artificial Sequence 5′ PCR primer 27 ataagaatgc ggccgccacc atgaaaaaag ggaaattagg agcc 44 28 33 DNA Artificial Sequence 3′ PCR primer 28 gcgccggatc cccgaagcag aagtcgttgt ggg 33 29 48 DNA Artificial Sequence 5′ PCR primer 29 ataagaatgc ggccgccacc atgagaaaac ttattttatg caatccta 48 30 33 DNA Artificial Sequence 3′ PCR primer 30 gcgccggatc ccagaacaac ggagttcttt tgg 33 31 46 DNA Artificial Sequence 5′ PCR primer 31 ataagaatgc ggccgccacc atgaataaaa aaaatctaac tatttg 46 32 32 DNA Artificial Sequence 3′ PCR primer 32 gcgccggatc ccagcgatag cttctggggt cc 32 33 42 DNA Artificial Sequence 5′ PCR primer 33 ataagaatgc ggccgccacc atgacactgg taccctatgt tg 42 34 35 DNA Artificial Sequence 3′ PCR primer 34 gcgccggatc ccagtgctac ttgtatcctt attag 35 35 45 DNA Artificial Sequence 5′ PCR primer 35 ataagaatgc ggccgccacc atgagctacc gtaaacgttc gactc 45 36 35 DNA Artificial Sequence 3′ PCR primer 36 gcgccggatc cccctcgttc ccccttgttt cggag 35 37 46 DNA Artificial Sequence 5′ PCR primer 37 ataagaatgc ggccgccacc atgtttgaga agttcactaa tagagc 46 38 34 DNA Artificial Sequence 3′ PCR primer 38 gcgccggtac cgtgattcca agtgagggct aggg 34 39 42 DNA Artificial Sequence 5′ PCR primer 39 ataagaatgc ggccgccacc atggtaaaga tcatatcaag tg 42 40 30 DNA Artificial Sequence 3′ PCR primer 40 gcgccggatc ccagcgtgct tattgataag 30 41 17 PRT Artificial Sequence B-cell epitope of ATP-binding cassette protein 41 Val His His Thr Leu Arg Glu Ser Tyr Lys Lys Gly Thr Pro Pro 1 5 10 15 Ser Thr 42 16 PRT Artificial Sequence B-cell epitope of ATP-binding cassette protein 42 Asn Leu Gln Lys Glu Ile Ser Thr Glu Glu Arg Gln Thr Lys Ala 1 5 10 15 Arg 43 9 PRT Artificial Sequence T-cell epitope of ATP-binding cassette protein 43 Trp Ile Ala Glu Tyr Val Ser Pro Val 1 5 44 12 PRT Artificial Sequence B-cell epitope of endopeptidase protein 44 Lys Gly Asn Asn Ser Ser Pro Arg Ser Pro Ala Pro 1 5 10 45 9 PRT Artificial Sequence B-cell epitope of endopeptidase protein 45 Gly Glu Asn Phe Gln Lys Asn Ser Ser 1 5 46 9 PRT Artificial Sequence T-cell epitope of endopeptidase protein 46 Leu Leu Ile Glu Asp Met Asp Leu Ile 1 5 47 9 PRT Artificial Sequence T-cell epitope of endopeptidase protein 47 Asn Leu Leu Ile Glu Asp Met Asp Leu 1 5 48 16 PRT Artificial Sequence B-cell epitope of protease protein 48 Thr Asp Leu Glu Gly Leu Glu Glu Asp His Lys Asp Ser Pro Trp 1 5 10 15 Glu 49 18 PRT Artificial Sequence B-cell epitope of protease protein 49 Ser Glu Asn Ala Lys Lys Ser Glu Glu Gln Thr Ser Pro Gln Glu 1 5 10 15 Thr Pro Glu 50 9 PRT Artificial Sequence T-cell epitope of protease protein 50 Tyr Leu Gly Asp Glu Ile Leu Glu Val 1 5 51 9 PRT Artificial Sequence T-cell epitope of protease protein 51 Tyr Leu Tyr Ser Leu Leu Ser Met Leu 1 5 52 8 PRT Artificial Sequence B-cell epitope of metalloprotease protein 52 Thr Thr Asn Arg Gln Lys Ala Leu 1 5 53 11 PRT Artificial Sequence B-cell epitope of metalloprotease protein 53 Val Asn Ser Ser Asn Ser Asn Arg Leu Arg Glu 1 5 10 54 9 PRT Artificial Sequence T-cell epitope of metalloprotease protein 54 Ser Val Leu Ser Arg Val Asn Tyr Val 1 5 55 9 PRT Artificial Sequence T-cell epitope of metalloprotease protein 55 Lys Leu Ser Ser Leu Ile Pro Gly Leu 1 5 56 9 PRT Artificial Sequence T-cell epitope of metalloprotease protein 56 Ile Leu Ile Gly His Lys Lys His Val 1 5 57 14 PRT Artificial Sequence B-cell epitope of CLP protease ATPase protein 57 Pro Pro Lys Gly Gly Arg Lys His Pro Asn Gln Glu Tyr Ile 5 10 58 14 PRT Artificial Sequence B-cell epitope of CLP protease ATPase protein 58 Ser Asp Asp Gln Ala Asp Leu Ser Gln Lys Thr Arg Asp His 1 5 10 59 9 PRT Artificial Sequence T-cell epitope of CLP protease ATPase protein 59 Lys Ile Leu Asp Val Pro Phe Thr Ile 1 5 60 9 PRT Artificial Sequence T-cell epitope of CLP protease ATPase protein 60 Leu Leu Gln Ala Ala Asp Tyr Asp Val 1 5 61 9 PRT Artificial Sequence B-cell epitope of CLP protease subunit protein 61 Gly Thr Lys Gly Lys Arg His Ala Leu 1 5 62 11 PRT Artificial Sequence B-cell epitope of CLP protease subunit protein 62 Ala Lys Glu Thr Asn Lys Asp Thr Ser Ser Thr 1 5 10 63 9 PRT Artificial Sequence T-cell epitope of CLP protease subunit protein 63 Ala Ile Tyr Asp Thr Ile Arg Phe Leu 1 5 64 19 PRT Artificial Sequence B-cell epitope of translycolase/transpeptidase protein 64 Asp Pro Thr Asn Tyr Lys Glu Tyr Phe Asn Asn Lys Glu Arg Ile 1 5 10 15 Glu His Thr Lys 65 17 PRT Artificial Sequence B-cell epitope of translycolase/transpeptidase protein 65 Lys Arg Leu Tyr Glu Glu Trp Asn Arg Ser Pro Lys Gln Gly Gly 1 5 10 15 Thr Arg 66 9 PRT Artificial Sequence T-cell epitope of translycolase/transpeptidase protein 66 Ala Leu Gly Gln His Glu Phe Cys Val 1 5 67 9 PRT Artificial Sequence T-cell epitope of translycolase/transpeptidase protein 67 Ile Leu Ala Thr Gly Ile Gln Met Val 1 5 68 26 PRT Artificial Sequence B-cell epitope of CLPc protease protein 68 Arg Asp Glu Glu Lys Lys Leu Arg Glu Arg Leu Gln Ser Met Lys 1 5 10 15 Gln Glu Trp Glu Asn His Lys Glu Glu His Gln 20 25 69 16 PRT Artificial Sequence B-cell epitope of CLPc protease protein 69 Ile Arg Arg Ser Arg Thr Gly Ile Lys Asp Pro Asn Arg Pro Thr 1 5 10 15 Gly 70 9 PRT Artificial Sequence T-cell epitope of CLPc protease protein 70 Phe Leu Phe Leu Gly Pro Thr Gly Val 1 5 71 9 PRT Artificial Sequence T-cell epitope of CLPc protease protein 71 Phe Leu Pro Asp Lys Ala Ile Asp Leu 1 5 72 13 PRT Artificial Sequence B-cell epitope of thioredoxin 72 Asn Ile Asp Glu Asn Ser Lys Pro Ala Glu Thr Tyr Glu 1 5 10 73 9 PRT Artificial Sequence B-cell epitope of thioredoxin 73 Asn Leu Ala Ala Glu Leu Pro His Val 1 5 74 9 PRT Artificial Sequence T-cell epitope of thioredoxin 74 Ile Leu Phe Lys Asp Gly Asn Glu Val 1 5

Claims (360)

1. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) a nucleic acid sequence set forth in any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed.
2. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed.
3. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) SEQ ID No: 1;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 1;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 1; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 2;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed.
4. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 1;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 2;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed.
5. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) SEQ ID No: 1;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 1;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 1; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 2;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
6. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 1;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 2;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
7. The vaccine of claim 5 or 6 wherein the second polypeptide is a heterologous signal peptide.
8. The vaccine of claim 5 or 6 wherein the second polypeptide has adjuvant activity.
9. The vaccine of any one of claims 3 to 8 wherein wherein each first nucleic acid is operatively linked to one or more expression control sequences.
10. A vaccine according to any one of claims 3 to 9, further comprising a second nucleic acid encoding an additional polypeptide which enhances the immune response to the polypeptide expressed by the first nucleic acid.
11. The vaccine of claim 10 wherein the second nucleic acid encodes an additional Chlamydia polypeptide.
12. A pharmaceutical composition comprising a vaccine according to any one of claims 3 to 11 and a pharmaceutically acceptable carrier.
13. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 1;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 2 or to the polypeptide encoded by SEQ ID NO: 1;
(iii) a polypeptide of SEQ ID NO: 2; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
14. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 1;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 2;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
15. The fusion protein of claim 13 or 14 wherein the second polypeptide is a heterologous signal peptide.
16. The fusion protein of claim 13 or 14 wherein the second polypeptide has adjuvant activity.
17. An antibody against the fusion protein of any one of claims 13 to 15.
18. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 1;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 2 or to the polypeptide encoded by SEQ ID NO: 1;
(iii) a polypeptide of SEQ ID NO: 2; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
19. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 1;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 2;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
20. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 1;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 2 or to the polypeptide encoded by SEQ ID NO: 1;
(iii) a polypeptide of SEQ ID NO: 2; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
21. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 1;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 2;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
22. The vaccine of claim 20 or 21 wherein the second polypeptide is a heterologous signal peptide.
23. The vaccine of claim 20 or 21 wherein the second polypeptide has adjuvant activity.
24. A vaccine according to any one of claims 18 to 23, further comprising an additional polypeptide which enhances the immune response to the first polypeptide.
25. The vaccine according to claim 24 wherein the additional polypeptide comprises a Chlamydia polypeptide.
26. A pharmaceutical composition comprising a vaccine according to any one of claims 18 to 25 and a pharmaceutically acceptable carrier.
27. A pharmaceutical composition comprising an antibody according to claim 17 and a pharmaceutically acceptable carrier.
28. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the vaccine of any one of claims 3 to 11 and 18 to 25.
29. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the composition of any one of claims 12, 26 and 27.
30. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the fusion protein of any one of claims 13 to 16.
31. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the antibody of claim 17.
32. A commercial package comprising at least one nucleic acid selected from any one of:
(i) SEQ ID No: 1;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 1;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 1; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 2;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed;
and instructions for use in eliciting an immunoprotective response in a mammal.
33. A commercial package comprising at least one nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 1;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 2;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
34. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 1;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 2 or to the polypeptide encoded by SEQ ID NO: 1;
(iii) a polypeptide of SEQ ID NO: 2; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii);
and instructions for use in eliciting an immunoprotective response in a mammal.
35. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 1;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 2;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii);
and instructions for use in eliciting an immunoprotective response in a mammal.
36. Expression plasmid pCACPNM213 as shown in FIG. 21.
37. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from:
(i) a nucleic acid encoding a polypeptide of any one of SEQ ID Nos: 41 to 43; and
(ii) a nucleic acid sequence as defined in (i) which has been modified to encode a modified conservatively substituted polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
38. A vaccine comprising a vaccine vector and at least one first polypeptide selected from:
(i) a polypeptide of any one of SEQ ID Nos: 41 to 43; and
(ii) a polypeptide as defined in (i) which has been modified by conservative substitution, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
39. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) SEQ ID No: 3;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 3;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 3; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 4;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed.
40. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 3;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 4;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed.
41. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) SEQ ID No: 3;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 3;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 3; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 4;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
42. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 3;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 4;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
43. The vaccine of claim 41 or 42 wherein the second polypeptide is a heterologous signal peptide.
44. The vaccine of claim 41 or 42 wherein the second polypeptide has adjuvant activity.
45. The vaccine of any one of claims 39 to 44 wherein wherein each first nucleic acid is operatively linked to one or more expression control sequences.
46. A vaccine according to any one of claims 39 to 45, further comprising a second nucleic acid encoding an additional polypeptide which enhances the immune response to the polypeptide expressed by the first nucleic acid.
47. The vaccine of claim 46 wherein the second nucleic acid encodes an additional Chlamydia polypeptide.
48. A pharmaceutical composition comprising a vaccine according to any one of claims 39 to 47 and a pharmaceutically acceptable carrier.
49. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 3;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 4 or to the polypeptide encoded by SEQ ID NO: 3;
(iii) a polypeptide of SEQ ID NO: 4; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
50. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 3;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 4;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
51. The fusion protein of claim 49 or 50 wherein the second polypeptide is a heterologous signal peptide.
52. The fusion protein of claim 49 or 50 wherein the second polypeptide has adjuvant activity.
53. An antibody against the fusion protein of any one of claims 49 to 51.
54. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 3;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 4 or to the polypeptide encoded by SEQ ID NO: 3;
(iii) a polypeptide of SEQ ID NO: 4; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
55. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 3;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 4;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
56. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 3;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 4 or to the polypeptide encoded by SEQ ID NO: 3;
(iii) a polypeptide of SEQ ID NO: 4; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
57. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 3;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 4;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
58. The vaccine of claim 56 or 57 wherein the second polypeptide is a heterologous signal peptide.
59. The vaccine of claim 56 or 57 wherein the second polypeptide has adjuvant activity.
60. A vaccine according to any one of claims 54 or 59, further comprising an additional polypeptide which enhances the immune response to the first polypeptide.
61. The vaccine according to claim 60 wherein the additional polypeptide comprises a Chlamydia polypeptide.
62. A pharmaceutical composition comprising a vaccine according to any one of claims 54 to 61 and a pharmaceutically acceptable carrier.
63. A pharmaceutical composition comprising an antibody according to claim 53 and a pharmaceutically acceptable carrier.
64. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the vaccine of any one of claims 39 to 47 and 54 to 61.
65. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the composition of any one of claims 48, 62 and 63.
66. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the fusion protein of any one of claims 49 to 52.
67. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the antibody of claim 53.
68. A commercial package comprising at least one nucleic acid selected from any one of:
(i) SEQ ID No: 3;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 3;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 3; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 4;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
69. A commercial package comprising at least one nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 3;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 4;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
70. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 3;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 4 or to the polypeptide encoded by SEQ ID NO: 3;
(iii) a polypeptide of SEQ ID NO: 4; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii); and
instructions for use in eliciting an immunoprotective response in a mammal.
71. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 3;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 4;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii);
and instructions for use in eliciting an immunoprotective response in a mammal.
72. Expression plasmid pCACPNM882 as shown in FIG. 22.
73. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) SEQ ID No: 5;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 5;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 5; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 6;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed.
74. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 5;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 6;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed.
75. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) SEQ ID No: 5;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 5;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 5; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 6;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
76. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 5;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 6;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
77. The vaccine of claim 75 or 76 wherein the second polypeptide is a heterologous signal peptide.
78. The vaccine of claim 75 or 76 wherein the second polypeptide has adjuvant activity.
79. The vaccine of any one of claims 73 to 78 wherein each first nucleic acid is operatively linked to one or more expression control sequences.
80. A vaccine according to any one of claims 73 to 79, further comprising a second nucleic acid encoding an additional polypeptide which enhances the immune response to the polypeptide expressed by the first nucleic acid.
81. The vaccine of claim 80 wherein the second nucleic acid encodes an additional Chlamydia polypeptide.
82. A pharmaceutical composition comprising a vaccine according to any one of claims 73 to 81 and a pharmaceutically acceptable carrier.
83. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 5;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 6 or to the polypeptide encoded by SEQ ID NO: 5;
(iii) a polypeptide of SEQ ID NO: 6; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
84. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 5;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 6;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
85. The fusion protein of claim 83 or 84 wherein the second polypeptide is a heterologous signal peptide.
86. The fusion protein of claim 83 or 84 wherein the second polypeptide has adjuvant activity.
87. An antibody against the fusion protein of any one of claims 83 to 85.
88. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 5;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 6 or to the polypeptide encoded by SEQ ID NO: 5;
(iii) a polypeptide of SEQ ID NO: 6; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
89. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 5;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 6;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
90. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 5;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 6 or to the polypeptide encoded by SEQ ID NO: 5;
(iii) a polypeptide of SEQ ID NO: 6; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
91. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 5;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 6;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
92. The vaccine of claim 90 or 91 wherein the second polypeptide is a heterologous signal peptide.
93. The vaccine of claim 90 or 91 wherein the second polypeptide has adjuvant activity.
94. A vaccine according to any one of claims 88 to 93, further comprising an additional polypeptide which enhances the immune response to the first polypeptide.
95. The vaccine according to claim 94 wherein the additional polypeptide comprises a Chlamydia polypeptide.
96. A pharmaceutical composition comprising a vaccine according to any one of claims 88 to 95 and a pharmaceutically acceptable carrier.
97. A pharmaceutical composition comprising an antibody according to claim 87 and a pharmaceutically acceptable carrier.
98. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the vaccine of any one of claims 73 to 81 and 88 to 95.
99. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the composition of any one of claims 82, 96 and 97.
100. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the fusion protein of any one of claims 83 to 86.
101. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the antibody of claim 87.
102. A commercial package comprising at least one nucleic acid selected from any one of:
(i) SEQ ID No: 5;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 5;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 5; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 6;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
103. A commercial package comprising at least one nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 5;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 6;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
104. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 5;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 6 or to the polypeptide encoded by SEQ ID NO: 5;
(iii) a polypeptide of SEQ ID NO: 6; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii); and
instructions for use in eliciting an immunoprotective response in a mammal.
105. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 5;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 6;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii); and
instructions for use in eliciting an immunoprotective response in a mammal.
106. Expression plasmid pCACPNM208 as shown in FIG. 23.
107. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from:
(i) a nucleic acid encoding a polypeptide of any one of SEQ ID Nos: 44 to 47; and
(ii) a nucleic acid sequence as defined in (i) which has been modified to encode a modified conservatively substituted polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
108. A vaccine comprising a vaccine vector and at least one first polypeptide selected from:
(i) a polypeptide of any one of SEQ ID Nos: 44 to 47; and
(ii) a polypeptide as defined in (i) which has been modified by conservative substitution, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
109. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) SEQ ID No: 7;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 7;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 7; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 8;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed.
110. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 7;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 8;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed.
111. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) SEQ ID No: 7;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 7;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 7; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 8;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
112. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 7;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 8;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
113. The vaccine of claim 111 or 112 wherein the second polypeptide is a heterologous signal peptide.
114. The vaccine of claim 111 or 112 wherein the second polypeptide has adjuvant activity.
115. The vaccine of any one of claims 109 to 114 wherein wherein each first nucleic acid is operatively linked to one or more expression control sequences.
116. A vaccine according to any one of claims 109 to 115, further comprising a second nucleic acid encoding an additional polypeptide which enhances the immune response to the polypeptide expressed by the first nucleic acid.
117. The vaccine of claim 116 wherein the second nucleic acid encodes an additional Chlamydia polypeptide.
118. A pharmaceutical composition comprising a vaccine according to any one of claims 109 to 117 and a pharmaceutically acceptable carrier.
119. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 7;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 8 or to the polypeptide encoded by SEQ ID NO: 7;
(iii) a polypeptide of SEQ ID NO: 8; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
120. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 7;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 8;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding. polypeptide of (i) or (ii).
121. The fusion protein of claim 119 or 120 wherein the second polypeptide is a heterologous signal peptide.
122. The fusion protein of claim 119 or 120 wherein the second polypeptide has adjuvant activity.
123. An antibody against the fusion protein of any one of claims 119 to 121.
124. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 7;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 8 or to the polypeptide encoded by SEQ ID NO: 7;
(iii) a polypeptide of SEQ ID NO: 8; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
125. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 7;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 8;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
126. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 7;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 8 or to the polypeptide encoded by SEQ ID NO: 7;
(iii) a polypeptide of SEQ ID NO: 8; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
127. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 7;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 8;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
128. The vaccine of claim 126 or 127 wherein the second polypeptide is a heterologous signal peptide.
129. The vaccine of claim 126 or 127 wherein the second polypeptide has adjuvant activity.
130. A vaccine according to any one of claims 124 to 129, further comprising an additional polypeptide which enhances the immune response to the first polypeptide.
131. The vaccine according to claim 130 wherein the additional polypeptide comprises a Chlamydia polypeptide.
132. A pharmaceutical composition comprising a vaccine according to any one of claims 124 to 131 and a pharmaceutically acceptable carrier.
133. A pharmaceutical composition comprising an antibody according to claim 123 and a pharmaceutically acceptable carrier.
134. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the vaccine of any one of claims 109 to 117 and 124 to 131.
135. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the composition of any one of claims 118, 132 and 133.
136. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the fusion protein of any one of claims 119 to 122.
137. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the antibody of claim 123.
138. A commercial package comprising at least one nucleic acid selected from any one of:
(i) SEQ ID No: 7;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 7;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 7; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 8;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
139. A commercial package comprising at least one nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 7;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 8;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
140. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 7;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 8 or to the polypeptide encoded by SEQ ID NO: 7;
(iii) a polypeptide of SEQ ID NO: 8; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii); and
instructions for use in eliciting an immunoprotective response in a mammal.
141. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 7;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 8;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii);
and instructions for use in eliciting an immunoprotective response in a mammal.
142. Expression plasmid pCACPNM1096 as shown in FIG. 24.
143. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from:
(i) a nucleic acid encoding a polypeptide of any one of SEQ ID Nos: 48 to 51; and
(ii) a nucleic acid sequence as defined in (i) which has been modified to encode a modified conservatively substituted polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
144. A vaccine comprising a vaccine vector and at least one first polypeptide selected from:
(i) a polypeptide of any one of SEQ ID Nos: 48 to 51; and
(ii) a polypeptide as defined in (i) which has been modified by conservative substitution, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
145. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) SEQ ID No: 9;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 9;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 9; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 10;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed.
146. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 9;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 10;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed.
147. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) SEQ ID No: 9;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 9;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 9; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 10;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
148. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 9;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 10;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
149. The vaccine of claim 147 or 148 wherein the second polypeptide is a heterologous signal peptide.
150. The vaccine of claim 147 or 148 wherein the second polypeptide has adjuvant activity.
151. The vaccine of any one of claims 145 to 150 wherein each first nucleic acid is operatively linked to one or more expression control sequences.
152. A vaccine according to any one of claims 145 to 151, further comprising a second nucleic acid encoding an additional polypeptide which enhances the immune response to the polypeptide expressed by the first nucleic acid.
153. The vaccine of claim 152 wherein the second nucleic acid encodes an additional Chlamydia polypeptide.
154. A pharmaceutical composition comprising a vaccine according to any one of claims 145 to 153 and a pharmaceutically acceptable carrier.
155. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 9;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 10 or to the polypeptide encoded by SEQ ID NO: 9;
(iii) a polypeptide of SEQ ID NO: 10; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
156. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 9;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 10;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
157. The fusion protein of claim 155 or 156 wherein the second polypeptide is a heterologous signal peptide.
158. The fusion protein of claim 155 or 156 wherein the second polypeptide has adjuvant activity.
159. An antibody against the fusion protein of any one of claims 155 to 157.
160. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 9;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 10 or to the polypeptide encoded by SEQ ID NO: 9;
(iii) a polypeptide of SEQ ID NO: 10; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
161. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 9;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 10;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
162. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 9;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 10 or to the polypeptide encoded by SEQ ID NO: 9;
(iii) a polypeptide of SEQ ID NO: 10; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
163. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 9;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 10;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
164. The vaccine of claim 162 or 163 wherein the second polypeptide is a heterologous signal peptide.
165. The vaccine of claim 162 or 163 wherein the second polypeptide has adjuvant activity.
166. A vaccine according to any one of claims 160 to 165, further comprising an additional polypeptide which enhances the immune response to the first polypeptide.
167. The vaccine according to claim 166 wherein the additional polypeptide comprises a Chlamydia polypeptide.
168. A pharmaceutical composition comprising a vaccine according to any one of claims 160 to 167 and a pharmaceutically acceptable carrier.
169. A pharmaceutical composition comprising an antibody according to claim 159 and a pharmaceutically acceptable carrier.
170. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the vaccine of any one of claims 145 to 153 and 160 to 167.
171. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the composition of any one of claims 154, 168 and 169.
172. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the fusion protein of any one of claims 155 to 158.
173. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the antibody of claim 159.
174. A commercial package comprising at least one nucleic acid selected from any one of:
(i) SEQ ID No: 9;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 9;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 9; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 10;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
175. A commercial package comprising at least one nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 9;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 10;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
176. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 9;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 10 or to the polypeptide encoded by SEQ ID NO: 9;
(iii) a polypeptide of SEQ ID NO: 10; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii); and
instructions for use in eliciting an immunoprotective response in a mammal.
177. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 9;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 10;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii); and
instructions for use in eliciting an immunoprotective response in a mammal.
178. Expression plasmid pCACPNM1097 as shown in FIG. 25.
179. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from:
(i) a nucleic acid encoding a polypeptide of any one of SEQ ID Nos: 52 to 56; and
(ii) a nucleic acid sequence as defined in (i) which has been modified to encode a modified conservatively substituted polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
180. A vaccine comprising a vaccine vector and at least one first polypeptide selected from:
(i) a polypeptide of any one of SEQ ID Nos: 52 to 56; and
(ii) a polypeptide as defined in (i) which has been modified by conservative substitution, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino-acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
181. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) SEQ ID No: 11;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 11;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 11; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 12;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed.
182. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 11;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 12;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed.
183. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) SEQ ID No: 11;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 11;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 11; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 12;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
184. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 11;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 12;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
185. The vaccine of claim 183 or 184 wherein the second polypeptide is a heterologous signal peptide.
186. The vaccine of claim 183 or 184 wherein the second polypeptide has-adjuvant activity.
187. The vaccine of any one of claims 181 to 186 wherein wherein each first nucleic acid is operatively linked to one or more expression control sequences.
188. A vaccine according to any one of claims 181 to 187, further comprising a second nucleic acid encoding an additional polypeptide which enhances the immune response to the polypeptide expressed by the first nucleic acid.
189. The vaccine of claim 188 wherein the second nucleic acid encodes an additional Chlamydia polypeptide.
190. A pharmaceutical composition comprising a vaccine according to any one of claims 181 to 189 and a pharmaceutically acceptable carrier.
191. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 11;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 12 or to the polypeptide encoded by SEQ ID NO: 11;
(iii) a polypeptide of SEQ ID NO: 12; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
192. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 11;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 12;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
193. The fusion protein of claim 191 or 192 wherein the second polypeptide is a heterologous signal peptide.
194. The fusion protein of claim 191 or 192 wherein the second polypeptide has adjuvant activity.
195. An antibody against the fusion protein of any one of claims 191 to 193.
196. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 11;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 12 or to the polypeptide encoded by SEQ ID NO: 11;
(iii) a polypeptide of SEQ ID NO: 12; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
197. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 11;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 12;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
198. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 11;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 12 or to the polypeptide encoded by SEQ ID NO: 11;
(iii) a polypeptide of SEQ ID NO: 12; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
199. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 11;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 12;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
200. The vaccine of claim 198 or 199 wherein the second polypeptide is a heterologous signal peptide.
201. The vaccine of claim 198 or 199 wherein the second polypeptide has adjuvant activity.
202. A vaccine according to any one of claims 196 to 201, further comprising an additional polypeptide which enhances the immune response to the first polypeptide.
203. The vaccine according to claim 202 wherein the additional polypeptide comprises a Chlamydia polypeptide.
204. A pharmaceutical composition comprising a vaccine according to any one of claims 196 to 203 and a pharmaceutically acceptable carrier.
205. A pharmaceutical composition comprising an antibody according to claim 195 and a pharmaceutically acceptable carrier.
206. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the vaccine of any one of claims 181 to 189 and 196 to 203.
207. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the composition of any one of claims 190, 204 and 205.
208. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the fusion protein of any one of claims 191 to 194.
209. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the antibody of claim 195.
210. A commercial package comprising at least one nucleic acid selected from any one of:
(i) SEQ ID No: 11;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 11;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 11; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 12;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
211. A commercial package comprising at least one nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 11;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 12;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
212. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 11;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 12 or to the polypeptide encoded by SEQ ID NO: 11;
(iii) a polypeptide of SEQ ID NO: 12; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii); and
instructions for use in eliciting an immunoprotective response in a mammal.
213. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 11;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 12;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii); and
instructions for use in eliciting an immunoprotective response in a mammal.
214. Expression plasmid pCACPNM908 as shown in FIG. 26.
215. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from:
(i) a nucleic acid encoding a polypeptide of any one of SEQ ID Nos: 57 to 60; and
(ii) a nucleic acid sequence as defined in (i) which has been modified to encode a modified conservatively substituted polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
216. A vaccine comprising a vaccine vector and at least one first polypeptide selected from:
(i) a polypeptide of any one of SEQ ID Nos: 57 to 60; and
(ii) a polypeptide as defined in (i) which has been modified by conservative substitution, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
217. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) SEQ ID No: 13;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 13;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 13; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 14;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed.
218. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 13;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 14;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed.
219. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) SEQ ID No: 13;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 13;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 13; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 14;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv); and
(b) a second polypeptide; wherein each first nucleic acid is capable of being expressed.
220. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 13;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 14;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
221. The vaccine of claim 219 or 220 wherein the second polypeptide is a heterologous signal peptide.
222. The vaccine of claim 219 or 220 wherein the second polypeptide has adjuvant activity.
223. The vaccine of any one of claims 217 to 222 wherein wherein each first nucleic acid is operatively linked to one or more expression control sequences.
224. A vaccine according to any one of claims 217 to 223, further comprising a second nucleic acid encoding an additional polypeptide which enhances the immune response to the polypeptide expressed by the first nucleic acid.
225. The vaccine of claim 224 wherein the second nucleic acid encodes an additional Chlamydia polypeptide.
226. A pharmaceutical composition comprising a vaccine according to any one of claims 217 to 225 and a pharmaceutically acceptable carrier.
227. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 13;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 14 or to the polypeptide encoded by SEQ ID NO: 13;
(iii) a polypeptide of SEQ ID NO: 14; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
228. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 13;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 14;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
229. The fusion protein of claim 227 or 228 wherein the second polypeptide is a heterologous signal peptide.
230. The fusion protein of claim 227 or 228 wherein the second polypeptide has adjuvant activity.
231. An antibody against the fusion protein of any one of claims 227 to 229.
232. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 13;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 14 or to the polypeptide encoded by SEQ ID NO: 13;
(iii) a polypeptide of SEQ ID NO: 14; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
233. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 13;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 14;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
234. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 13;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 14 or to the polypeptide encoded by SEQ ID NO: 13;
(iii) a polypeptide of SEQ ID NO: 14; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
235. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 13;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 14;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
236. The vaccine of claim 234 or 235 wherein the second polypeptide is a heterologous signal peptide.
237. The vaccine of claim 234 or 235 wherein the second polypeptide has adjuvant activity.
238. A vaccine according to any one of claims 232 to 237, further comprising an additional polypeptide which enhances the immune response to the first polypeptide.
239. The vaccine according to claim 238 wherein the additional polypeptide comprises a Chlamydia polypeptide.
240. A pharmaceutical composition comprising a vaccine according to any one of claims 232 to 239 and a pharmaceutically acceptable carrier.
241. A pharmaceutical composition comprising an antibody according to claim 231 and a pharmaceutically acceptable carrier.
242. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the vaccine of any one of claims 217 to 225 and 232 to 239.
243. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the composition of any one of claims 226, 240 and 241.
244. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the fusion protein of any one of claims 227 to 230.
245. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the antibody of claim 231.
246. A commercial package comprising at least one nucleic acid selected from any one of:
(i) SEQ ID No: 13;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 13;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 13; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 14;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
247. A commercial package comprising at least one nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 13;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 14;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
248. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 13;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 14 or to the polypeptide encoded by SEQ ID NO: 13;
(iii) a polypeptide of SEQ ID NO: 14; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii); and
instructions for use in eliciting an immunoprotective response in a mammal.
249. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 13;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 14;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii); and
instructions for use in eliciting an immunoprotective response in a mammal.
250. Expression plasmid pCACPNM909 as shown in FIG. 27.
251. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from:
(i) a nucleic acid encoding a polypeptide of any one of SEQ ID Nos: 61 to 63; and
(ii) a nucleic acid sequence as defined in (i) which has been modified to encode a modified conservatively substituted polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
252. A vaccine comprising a vaccine vector and at least one first polypeptide selected from:
(i) a polypeptide of any one of SEQ ID Nos: 61 to 63; and
(ii) a polypeptide as defined in (i) which has been modified by conservative substitution, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
253. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) SEQ ID No: 15;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 15;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 15; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 16;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed.
254. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 15;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 16;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed.
255. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) SEQ ID No: 15;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 15;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 15; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 16;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
256. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 15;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 16;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
257. The vaccine of claim 255 or 256 wherein the second polypeptide is a heterologous signal peptide.
258. The vaccine of claim 255 or 256 wherein the second polypeptide has adjuvant activity.
259. The vaccine of any one of claims 253 to 258 wherein wherein each first nucleic acid is operatively linked to one or more expression control sequences.
260. A vaccine according to any one of claims 253 to 259, further comprising a second nucleic acid encoding an additional polypeptide which enhances the immune response to the polypeptide expressed by the first nucleic acid.
261. The vaccine of claim 260 wherein the second nucleic acid encodes an additional Chlamydia polypeptide.
262. A pharmaceutical composition comprising a vaccine according to any one of claims 253 to 261 and a pharmaceutically acceptable carrier.
263. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 15;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 16 or to the polypeptide encoded by SEQ ID NO: 15;
(iii) a polypeptide of SEQ ID NO: 16; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
264. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 15;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 16;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
265. The fusion protein of claim 263 or 264 wherein the second polypeptide is a heterologous signal peptide.
266. The fusion protein of claim 263 or 264 wherein the second polypeptide has adjuvant activity.
267. An antibody against the fusion protein of any one of claims 263 to 265.
268. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 15;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 16 or to the polypeptide encoded by SEQ ID NO: 15;
(iii) a polypeptide of SEQ ID NO: 16; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
269. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 15;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 16;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
270. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 15;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 16 or to the polypeptide encoded by SEQ ID NO: 15;
(iii) a polypeptide of SEQ ID NO: 16; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
271. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 15;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 16;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
272. The vaccine of claim 270 or 271 wherein the second polypeptide is a heterologous signal peptide.
273. The vaccine of claim 270 or 271 wherein the second polypeptide has adjuvant activity.
274. A vaccine according to any one of claims 268 to 273, further comprising an additional polypeptide which enhances the immune response to the first polypeptide.
275. The vaccine according to claim 274 wherein the additional polypeptide comprises a Chlamydia polypeptide.
276. A pharmaceutical composition comprising a vaccine according to any one of claims 268 to 275 and a pharmaceutically acceptable carrier.
277. A pharmaceutical composition comprising an antibody according to claim 267 and a pharmaceutically acceptable carrier.
278. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the vaccine of any one of claims 253 to 261 and 268 to 275.
279. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the composition of any one of claims 262, 276 and 277.
280. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the fusion protein of any one of claims 263 to 266.
281. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the antibody of claim 267.
282. A commercial package comprising at least one nucleic acid selected from any one of:
(i) SEQ ID No: 15;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 15;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 15; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 16;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
283. A commercial package comprising at least one nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 15;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 16;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
284. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 15;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 16 or to the polypeptide encoded by SEQ ID NO: 15;
(iii) a polypeptide of SEQ ID NO: 16; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii); and
instructions for use in eliciting an immunoprotective response in a mammal.
285. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 15;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 16;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii); and
instructions for use in eliciting an immunoprotective response in a mammal.
286. Expression plasmid pCACPNM440 as shown in FIG. 28.
287. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from:
(i) a nucleic acid encoding a polypeptide of any one of SEQ ID Nos: 64 to 67; and
(ii) a nucleic acid sequence as defined in (i) which has been modified to encode a modified conservatively substituted polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
288. A vaccine comprising a vaccine vector and at least one first polypeptide selected from:
(i) a polypeptide of any one of SEQ ID Nos: 64 to 67; and
(ii) a polypeptide as defined in (i) which has been modified by conservative substitution, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
289. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) SEQ ID No: 17;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 17;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 17; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 18;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed.
290. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 17;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 18;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed.
291. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) SEQ ID No: 17;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 17;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 17; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 18;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
292. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 17;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 18;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
293. The vaccine of claim 291 or 292 wherein the second polypeptide is a heterologous signal peptide.
294. The vaccine of claim 291 or 292 wherein the second polypeptide has adjuvant activity.
295. The vaccine of any one of claims 289 to 294 wherein wherein each first nucleic acid is operatively linked to one or more expression control sequences.
296. A vaccine according to any one of claims 289 to 295, further comprising a second nucleic acid encoding an additional polypeptide which enhances the immune response to the polypeptide expressed by the first nucleic acid.
297. The vaccine of claim 296 wherein the second nucleic acid encodes an additional Chlamydia polypeptide.
298. A pharmaceutical composition comprising a vaccine according to any one of claims 289 to 297 and a pharmaceutically acceptable carrier.
299. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 17;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 18 or to the polypeptide encoded by SEQ ID NO: 17;
(iii) a polypeptide of SEQ ID NO: 18; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
300. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 17;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 18;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
301. The fusion protein of claim 299 or 300 wherein the second polypeptide is a heterologous signal peptide.
302. The fusion protein of claim 299 or 300 wherein the second polypeptide has adjuvant activity.
303. An antibody against the fusion protein of any one of claims 299 to 301.
304. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 17;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 18 or to the polypeptide encoded by SEQ ID NO: 17;
(iii) a polypeptide of SEQ ID NO: 18; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
305. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 17;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 18;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
306. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 17;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 18 or to the polypeptide encoded by SEQ ID NO: 17;
(iii) a polypeptide of SEQ ID NO: 18; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
307. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 17;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 18;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
308. The vaccine of claim 306 or 307 wherein the second polypeptide is a heterologous signal peptide.
309. The vaccine of claim 306 or 307 wherein the second polypeptide has adjuvant activity.
310. A vaccine according to any one of claims 304 to 309, further comprising an additional polypeptide which enhances the immune response to the first polypeptide.
311. The vaccine according to claim 310 wherein the additional polypeptide comprises a Chlamydia polypeptide.
312. A pharmaceutical composition comprising a vaccine according to any one of claims 304 to 311 and a pharmaceutically acceptable carrier.
313. A pharmaceutical composition comprising an antibody according to claim 303 and a pharmaceutically acceptable carrier.
314. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the vaccine of any one of claims 289 to 297 and 304 to 311.
315. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the composition of any one of claims 298, 312 and 313.
316. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the fusion protein of any one of claims 299 to 302.
317. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the antibody of claim 303.
318. A commercial package comprising at least one nucleic acid selected from any one of:
(i) SEQ ID No: 17;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 17;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 17; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 18;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
319. A commercial package comprising at least one nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 17;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 18;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
320. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 17;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 18 or to the polypeptide encoded by SEQ ID NO: 17;
(iii) a polypeptide of SEQ ID NO: 18; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii);
and instructions for use in eliciting an immunoprotective response in a mammal.
321. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 17;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 18;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii);
and instructions for use in eliciting an immunoprotective response in a mammal.
322. Expression plasmid pCACPNM459 as shown in FIG. 29.
323. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from:
(i) a nucleic acid encoding a polypeptide of any one of SEQ ID Nos: 68 to 71; and
(ii) a nucleic acid sequence as defined in (i) which has been modified to encode a modified conservatively substituted polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
324. A vaccine comprising a vaccine vector and at least one first polypeptide selected from:
(i) a polypeptide of any one of SEQ ID Nos: 68 to 71; and
(ii) a polypeptide as defined in (i) which has been modified by conservative substitution, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
325. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) SEQ ID No: 19;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 19;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 19; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 20;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed.
326. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 19;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 20;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed.
327. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) SEQ ID No: 19;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 19;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 19; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 20;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
328. A vaccine comprising a vaccine vector and at least one first nucleic acid encoding a fusion protein, wherein the fusion protein comprises:
(a) a first polypeptide encoded by a nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 19;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 20;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii); and
(b) a second polypeptide;
wherein each first nucleic acid is capable of being expressed.
329. The vaccine of claim 327 or 328 wherein the second polypeptide is a heterologous signal peptide.
330. The vaccine of claim 327 or 328 wherein the second polypeptide has adjuvant activity.
331. The vaccine of any one of claims 325 to 330 wherein wherein each first nucleic acid is operatively linked to one or more expression control sequences.
332. A vaccine according to any one of claims 325 to 331, further comprising a second nucleic acid encoding an additional polypeptide which enhances the immune response to the polypeptide expressed by the first nucleic acid.
333. The vaccine of claim 332 wherein the second nucleic acid encodes an additional Chlamydia polypeptide.
334. A pharmaceutical composition comprising a vaccine according to any one of claims 325 to 333 and a pharmaceutically acceptable carrier.
335. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 19;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 20 or to the polypeptide encoded by SEQ ID NO: 19;
(iii) a polypeptide of SEQ ID NO: 20; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
336. A fusion protein comprising a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 19;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 20;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
337. The fusion protein of claim 335 or 336 wherein the second polypeptide is a heterologous signal peptide.
338. The fusion protein of claim 335 or 336 wherein the second polypeptide has adjuvant activity.
339. An antibody against the fusion protein of any one of claims 335 to 337.
340. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 19;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 20 or to the polypeptide encoded by SEQ ID NO: 19;
(iii) a polypeptide of SEQ ID NO: 20; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
341. A vaccine comprising at least one first polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 19;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 20;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
342. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 19;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 20 or to the polypeptide encoded by SEQ ID NO: 19;
(iii) a polypeptide of SEQ ID NO: 20; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii).
343. A vaccine comprising at least one fusion protein, wherein the fusion protein comprises a first and a second polypeptide, wherein the first polypeptide is selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 19;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 20;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii).
344. The vaccine of claim 342 or 343 wherein the second polypeptide is a heterologous signal peptide.
345. The vaccine of claim 342 or 343 wherein the second polypeptide has adjuvant activity.
346. A vaccine according to any one of claims 340 to 345, further comprising an additional polypeptide which enhances the immune response to the first polypeptide.
347. The vaccine according to claim 346 wherein the additional polypeptide comprises a Chlamydia polypeptide.
348. A pharmaceutical composition comprising a vaccine according to any one of claims 340 to 347 and a pharmaceutically acceptable carrier.
349. A pharmaceutical composition comprising an antibody according to claim 339 and a pharmaceutically acceptable carrier.
350. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the vaccine of any one of claims 325 to 333 and 340 to 347.
351. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the composition of any one of claims 334, 348 and 349.
352. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the fusion protein of any one of claims 335 to 338.
353. A method for preventing or treating Chlamydia infection comprising administering to a mammal an effective amount of the antibody of claim 339.
354. A commercial package comprising at least one nucleic acid selected from any one of:
(i) SEQ ID No: 19;
(ii) a nucleic acid sequence which encodes a polypeptide encoded by SEQ ID No: 19;
(iii) a nucleic acid sequence which encodes a polypeptide which is at least 75% identical in amino acid sequence to the polypeptide encoded by SEQ ID No: 19; and
(iv) a nucleic acid sequence which encodes a polypeptide whose sequence is set forth in SEQ ID No: 20;
(v) a nucleic acid sequence as defined in (i), (ii) or (iv), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iv);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
355. A commercial package comprising at least one nucleic acid selected from any one of:
(i) a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 19;
(ii) a nucleic acid sequence which encodes an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 20;
(iii) a nucleic acid sequence as defined in (i) or (ii), which has been modified to encode a modified polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding fragment of (i) or (ii);
wherein each first nucleic acid is capable of being expressed; and
instructions for use in eliciting an immunoprotective response in a mammal.
356. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by SEQ ID NO: 19;
(ii) a polypeptide which is at least 75% identical in amino acid sequence to SEQ ID NO: 20 or to the polypeptide encoded by SEQ ID NO: 19;
(iii) a polypeptide of SEQ ID NO: 20; and
(iv) a polypeptide as defined in (i), (ii) or (iii) which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i), (ii) or (iii); and
instructions for use in eliciting an immunoprotective response in a mammal.
357. A commercial package comprising at least one polypeptide selected from any one of:
(i) a polypeptide encoded by a nucleic acid sequence comprising at least 36 consecutive nucleotides from SEQ ID NO: 19;
(ii) a polypeptide which is an immunogenic fragment comprising at least 12 consecutive amino acids from SEQ ID No: 20;
(iii) a polypeptide as defined in (i) or (ii), which has been modified without loss of immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide of (i) or (ii); and
instructions for use in eliciting an immunoprotective response in a mammal.
358. Expression plasmid pCACPNM708 as shown in FIG. 30.
359. A vaccine comprising a vaccine vector and at least one first nucleic acid selected from:
(i) a nucleic acid encoding a polypeptide of any one of SEQ ID Nos: 72 to 74; and
(ii) a nucleic acid sequence as defined in (i) which has been modified to encode a modified conservatively substituted polypeptide, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
360. A vaccine comprising a vaccine vector and at least one first polypeptide selected from:
(i) a polypeptide of any one of SEQ ID Nos: 72 to 74; and
(ii) a polypeptide as defined in (i) which has been modified by conservative substitution, wherein the modified polypeptide retains immunogenicity and is at least 75% identical in amino acid sequence to the corresponding polypeptide encoded by the nucleic acid of (i).
US10/275,652 2000-05-08 2001-05-08 Chlamydia antigens and corresponding dna fragments and uses thereof Abandoned US20040254130A1 (en)

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Application Number Priority Date Filing Date Title
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Applications Claiming Priority (10)

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US20267200P 2000-05-08 2000-05-08
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US7297341B1 (en) 1998-12-23 2007-11-20 Sanofi Pasteur Limited Chlamydia antigens and corresponding DNA fragments and uses thereof
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US7736873B2 (en) 1998-12-01 2010-06-15 Sanofi Pasteur Limited Chlamydia polypeptides and corresponding DNA fragments and uses thereof
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US7297341B1 (en) 1998-12-23 2007-11-20 Sanofi Pasteur Limited Chlamydia antigens and corresponding DNA fragments and uses thereof
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US20080113933A1 (en) * 1999-03-12 2008-05-15 Murdin Andrew D Chlamydia 60 kDa CRMP antigens and vaccine uses
US7285276B2 (en) 1999-03-12 2007-10-23 Sanofi Pasteur Limited Chlamydia antigens and corresponding DNA fragments and uses thereof
US7183402B2 (en) 1999-03-12 2007-02-27 Sanofi Pasteur Limited Chlamydia antigens and corresponding DNA fragments and uses thereof
US7629327B2 (en) 1999-03-12 2009-12-08 Sanofi Pasteur Limited Chlamydia 60 Kda CRMP antigens and vaccine uses
US20050186266A1 (en) * 1999-03-12 2005-08-25 Aventis Pasteur Limited Chlamydia antigens and corresponding DNA fragments and uses thereof
US7595058B2 (en) 1999-05-03 2009-09-29 Sanofi Pasteur Limited Chlamydia antigens and vaccine uses of the protein
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US20050220805A1 (en) * 1999-09-20 2005-10-06 Aventis Pasteur Limited Chlamydia antigens and corresponding DNA fragments and uses thereof
US7662391B2 (en) 1999-09-20 2010-02-16 Sanofi Pasteur Limited Chlamydia outer membrane protein (OMP) and vaccine uses of the protein
US20050002944A1 (en) * 1999-09-20 2005-01-06 Aventis Pasteur Limited Chlamydia antigens and corresponding DNA fragments and uses thereof
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